Factor VIII activation by factor VIIa analog (V158D/E296V/M298Q) in tissue factor-independent mechanisms

2011 ◽  
Vol 106 (10) ◽  
pp. 665-674 ◽  
Author(s):  
Kenichi Ogiwara ◽  
Midori Shima ◽  
Keiji Nogami
Keyword(s):  
Tissue Factor ◽  
Catalytic Domain ◽  
Factor Viia ◽  
Peak Level ◽  
Activated Platelets ◽  
Fviii Activity ◽  
Rapid Inactivation ◽  
Von Willebrand ◽  
A2 Domain ◽  
Willebrand Factor

SummaryFactor (F)VIIa with tissue factor (TF) is a primary trigger of blood coagulation. The recombinant (r)FVIIa analog, NN1731 (V158D/E296V/ M298Q) containing a thrombin/FIXa-mimicking catalytic domain, is ~30-fold more effective on activated platelets without TF, but ~1.2-fold with TF, than rFVIIa for FX activation. We have recently demonstrated the FVIIa/TF-dependent FVIII activation in the early coagulation phase. We assessed the action of NN1731 on FVIII activation. NN1731/TF increased FVIII activity ~2.9-fold within 30 seconds, followed by rapid inactivation, and was slightly more active than rFVIIa/TF. NN1731-catalysed activation, however, was enhanced ~6-fold at 5 minutes (min), and its peak level persisted for ~30 min. NN1731/TF proteolysed FVIII at Arg740, Arg372, and Arg336, similar to rFVIIa/TF, but cleavage by NN1731 alone was much slower at Arg336 than at Arg740 and Arg372. The Km and Vmax for NN1731/TF-catalysed activation were ~1.8-fold lower and ~2.3-fold greater than rFVIIa/TF. The Km for NN1731 alone was ~1.3-fold lower than rFVIIa, whilst the Vmax was ~7.9-fold greater, indicating that the efficiency of FVIII activation by NN1731 and NN1731/TF was ~11- and ~4-fold greater, respectively, than equivalent reactions with rFVIIa. In SPR-based assays, NN1731 bound to FVIII and the heavy chain (Kd; 0.62 and 1.9 nM) with ~1.4- and ~3.1-fold higher affinity than rFVIIa, and the A2 domain contributed to this increase. Von Willebrand factor moderated NN1731-catalysed activation more significantly than NN1731/TF. In conclusion, NN1731 was a greater potential than rFVIIa in up-regulating FVIII activity, and the TF-independent FVIII activation might represent a potential extra mode of its enhanced haemostatic effect.

Mechanisms of Factor VIII Activation by Recombinant Factor VIIa Analog through Tissue Factor-Independent Manner

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1028-1028
Author(s):  
Kenichi Ogiwara ◽  
Keiji Nogami ◽  
Tetsuhiro Soeda ◽  
Tomoko Matsumoto ◽  
Katsumi Nishiya ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Blood Coagulation ◽  
Factor Viia ◽  
Physiological Role ◽  
Peak Level ◽  
Factor X ◽  
Independent Manner ◽  
Hemostatic Effect ◽  
Dependent Change ◽  
Fviii Activity

Abstract Factor VIIa (FVIIa), complexed with tissue factor (TF), is a trigger of blood coagulation. Analog of recombinant FVIIa (rFVIIa), NN1731 (V158D/E296V/M298Q) possesses a greater hemostatic effect than rFVIIa and has been expected in clinical application. Factor X activation rate of NN1731 compared to rFVIIa was 1.2-fold in the presence of TF (TF(+)), and was 30-fold on activated platelets in its absence (TF(−))(Allen, Arterioscler Thromb Vasc Biol.2007; 27: 683). This TF-independent mechanism likely attributes to excellent effects by NN1731. More recently, we reported the physiological role of FVIIa/TF-dependent FVIII activation in the early phase of blood coagulation. Therefore, we were tempted to investigate the action of NN1731 in FVIII activation. Time-dependent change in FVIII activity after the addition of rFVIIa/NN1731 was examined by one-stage clotting assay under the presence of phospholipids (PS:PC:PE=1:6:3), CaCl2 and TF(+)/TF(−). NN1731 raised FVIII activity up to peak level rapidly within 30 sec (TF(+)), following by inactivation. Peak level of FVIII activity by NN1731 in TF(−) reached to the same peak level of that in TF(+) within 5 min, and this peak level persisted for ~30 min. Whilst, peak FVIII level by rFVIIa in TF(−) showed only ~35% of that in TF(+) even at 30 min. FVIII activating rate of NN1731 was observed to be 1.2-fold (TF(+)) and 3.8-fold (TF(−)) of rFVIIa-catalyzed activation. Kinetics by the Xa generation assay showed the Km values of NN1731 in FVIII activation were ~1.5-fold lower than those of rFVIIa (NN1731/rFVIIa; TF(+) 27.3/49.2 nM and TF(−) 50.5/68.1 nM). Vmax values of NN1731 in FVIII activation, however, showed the obvious difference between TF(+) (2.3-fold; NN1731/rFVIIa 70.0/30.4 nM•min−1) and TF(−) (7.9-fold; 92.5/11.7 nM•min−1), compared to rFVIIa. Inactivation of FVIIIa by NN1731 was somewhat faster than that by rFVIIa. FVIII cleavages by NN1731 were analyzed using SDS-PAGE/Western blotting. The heavy chain of FVIII was proteolyzed at Arg740 (A2-B junction), Arg372 (A1-A2 junction) and Arg336 (within the A1), faster by NN1731 than by rFVIIa. These predominant cleavages by NN1731 were more evident in TF(−). However, little cleavage of the light chain of FVIII was observed by both proteases. FVIII cleavages were correlated with the observations of FVIII activation and/or inactivation. To further localize the binding region for NN1731, we evaluated the interactions between FVIII and Glu-Gly-Arg-active site modified (EGR-) NN1731, lacking enzymatic activity, in a surface plasmon resonance-based assay. The Kd value of EGR-NN1731 with FVIII was similar to that of EGR-rFVIIa (6.3 and 7.8 nM, respectively). Binding was particularly evident with the A2, A3, and C2 domains, whilst the A1 domain failed to bind, similar to the results obtained by rFVIIa. We demonstrated that NN1731 possesses higher potential as an activator for up-regulation of FVIII activity than rFVIIa. Furthermore, catalytic activity of NN1731 in TF(−), rather than binding affinity, likely attributes to this potential of its analog. We concluded that the analog has another novel mechanism in its potent hemostatic effect through FVIII activation in TF-independent manner.

Factor VIII Inhibitor Antibodies with C2 Domain Specificity Are Less inhibitory to Factor VIII Complexed with von Willebrand Factor

1996 ◽  
Vol 76 (05) ◽  
pp. 749-754 ◽  
Author(s):  
Suzuki Suzuki ◽  
Morio Arai ◽  
Kagehiro Amano ◽  
Kazuhiko Kagawa ◽  
Katsuyuki Fukutake
Keyword(s):  
Complex Formation ◽  
Factor Viii ◽  
Von Willebrand Factor ◽  
Domain Specificity ◽  
Factor Viii Inhibitor ◽  
C2 Domain ◽  
Recombinant Fviii ◽  
Von Willebrand ◽  
A2 Domain ◽  
Willebrand Factor

SummaryIn order to clarify the potential role of von Willebrand factor (vWf) in attenuating the inactivation of factor VIII (fVIII) by those antibodies with C2 domain specificity, we investigated a panel of 14 human antibodies to fVIII. Immunoblotting analysis localized light chain (C2 domain) epitopes for four cases, heavy chain (A2 domain) epitopes in five cases, while the remaining five cases were both light and heavy chains. The inhibitor titer was considerably higher for Kogenate, a recombinant fVIII concentrate, than for Haemate P, a fVIII/vWf complex concentrate, in all inhibitor plasmas that had C2 domain specificity. In five inhibitor plasmas with A2 domain specificity and in five with both A2 and C2 domain specificities, Kogenate gave titers similar to or lower than those with Haemate P. The inhibitory effect of IgG of each inhibitor plasma was then compared with recombinant fVIII and its complex with vWf. When compared to the other 10 inhibitor IgGs, IgG concentration, which inhibited 50% of fVIII activity (IC50), was remarkably higher for the fVIII/vWf complex than for fVIII in all the inhibitor IgGs that had C2 domain reactivity. Competition of inhibitor IgG and vWf for fVIII binding was observed in an ELISA system. In 10 inhibitors that had C2 domain reactivity, the dose dependent inhibition of fVIII-vWf complex formation was observed, while, in the group of inhibitors with A2 domain specificity, there was no inhibition of the complex formation except one case. We conclude that a subset of fVIII inhibitors, those that bind to C2 domain determinants, are less inhibitory to fVIII when it is complexed with vWf that binds to overlapping region in the C2 domain.

The Basis for the Increase in Factor VIII Procoagulant Activity During Exercise

1983 ◽  
Vol 49 (01) ◽  
pp. 053-057 ◽  
Author(s):  
Robert G Kopitsky ◽  
Mary Ellen P Switzer ◽  
R Sanders Williams ◽  
Patrick A McKee
Keyword(s):  
Factor Viii ◽  
Acute Exercise ◽  
Post Exercise ◽  
Fviii Activity ◽  
Plasma Factor ◽  
The Subject ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Healthy Males ◽  
Related Increase

SummaryWe studied the effect of acute exercise on the ability of thrombin to activate plasma factor VIII (FVIII) activity in 20 healthy males. The subject showed an average exercise-related increase in FVIII activity of 54.5±8.2% over pre-exercise FVIII activity (p<0.001). When exposed to the same concentration of thrombin, post-exercise FVIII activity showed greater enhancement than pre-exercise FVIII activity: 157.1±12.8% increase in activity versus 117.3±9.9%, respectively (p<0.01). The degree of the potentiated thrombin effect in post-exercise samples relative to pre-exercise samples was linearly correlated with the degree of the exercise-related increase in FVIII activity. Taken together with our previous observations that the extent of thrombin enhancement of FVIII activity varies inversely with the mole ratio of FVIII/von Willebrand factor subunits to thrombin, these findings imply that release of FVIII does not occur during exercise, and that the exercise-related increase in FVIII activity results primarily, if not completely, from activation of already circulating but inactive FVIII.

scholarly journals The ADAMTS13 metalloprotease domain: roles of subsites in enzyme activity and specificity

Blood ◽  
2010 ◽  
Vol 116 (16) ◽  
pp. 3064-3072 ◽  
Author(s):  
Rens de Groot ◽  
David A. Lane ◽  
James T. B. Crawley
Keyword(s):  
Single Point ◽  
Minor Importance ◽  
Variable Regions ◽  
Cleavage Efficiency ◽  
Scissile Bond ◽  
A Minor ◽  
Von Willebrand ◽  
A2 Domain ◽  
Willebrand Factor

Abstract ADAMTS13 modulates von Willebrand factor (VWF) platelet-tethering function by proteolysis of the Tyr1605-Met1606 bond in the VWF A2 domain. To examine the role of the metalloprotease domain of ADAMTS13 in scissile bond specificity, we identified 3 variable regions (VR1, -2, and -3) in the ADAMTS family metalloprotease domain that flank the active site, which might be important for specificity. Eight composite sequence swaps (to residues in ADAMTS1 or ADAMTS2) and 18 single-point mutants were generated in these VRs and expressed. Swapping VR1 (E184-R193) of ADAMTS13 with that of ADAMTS1 or ADAMTS2 abolished/severely impaired ADAMTS13 function. Kinetic analysis of VR1 point mutants using VWF115 as a short substrate revealed reduced proteolytic function (kcat/Km reduced by 2- to 10-fold) as a result of D187A, R190A, and R193A substitutions. Analysis of VR2 (F216-V220) revealed a minor importance of this region. Mutants of VR3 (G236-A261) proteolysed wild-type VWF115 normally. However, using either short or full-length VWF substrates containing the P1′ M1606A mutation, we identified residues within VR3 (D252-P256) that influence P1′ amino acid specificity, we hypothesize, by shaping the S1′ pocket. It is concluded that 2 subsites, D187-R193 and D252-P256, in the metalloprotease domain play an important role in cleavage efficiency and site specificity.

Transcriptional Expression of Tissue Factor Pathway Inhibitor, Thrombomodulin and von Willebrand Factor in Normal Human Tissues

1999 ◽  
Vol 82 (09) ◽  
pp. 1047-1052 ◽  
Author(s):  
M. N. Kuppuswamy ◽  
A. N. Manepalli ◽  
S. P. Bajaj ◽  
M. S. Bajaj
Keyword(s):  
Skeletal Muscle ◽  
Tissue Factor ◽  
Von Willebrand Factor ◽  
Tissue Factor Pathway Inhibitor ◽  
Northern Blotting ◽  
Transcriptional Expression ◽  
Microvascular Endothelium ◽  
Tissue Factor Pathway ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryUnder normal physiologic conditions, tissue factor pathway inhibitor (TFPI) is synthesized primarily by the microvascular endothelium. Using Northern blotting, we studied its transcriptional expression in different organs and compared it with the expression of two other endothelial specific proteins, namely thrombomodulin (TM) and von Willebrand factor (vWF). The order of mRNA expression for each protein was: TFPI–placenta>lung>liver>kidney>heart>skeletal muscle≥pancreas>brain; TM–heart>pancreas>lung>skeletal muscle>kidney≥liver>placenta>brain; and vWF–heart>skeletal muscle>pancreas>lung≥kidney>placenta>brain>liver. Notably, heart expressed TM and vWF mRNA in large amounts and only small amounts of TFPI whereas lung expressed all three mRNAs in significant amounts. Placenta, on the contrary, expressed large amounts of TFPI but only small amounts of TM and vWF mRNAs. Brain by this technique was found to express undetectable amounts of TFPI and TM mRNAs but small amounts of vWF mRNA. The expression of TFPI mRNA in the brain was however detected by RT/PCR and the antigen was localized to the endothelium of microvessels as well as to the astrocytes and oligodendrocytes. Since ultimate expression of proteins is linked to the expression of their mRNAs, our data support a concept that vascular endothelium is made up of phenotypically diverse groups of cells and that endothelial cells of different vascular beds express specific sets of genes that enable them to carry out tissue-specific functions. Importantly, since astrocytes are known to express tissue factor, the TFPI expression by these cells may control coagulation in their microenvironment and their response to injury and inflammation.

scholarly journals Long-ranged Protein-glycan Interactions Stabilize von Willebrand Factor A2 Domain from Mechanical Unfolding

2018 ◽  
Vol 8 (1) ◽  
Author(s):  
Chuqiao Dong ◽  
Jumin Lee ◽  
Seonghoon Kim ◽  
Whitney Lai ◽  
Edmund B. Webb ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Von Willebrand ◽  
A2 Domain ◽  
Willebrand Factor

scholarly journals Factor VIII Inhibitors: Von Willebrand Factor Makes A Difference In Vitro and In Vivo

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 709-709
Author(s):  
Qizhen Shi ◽  
Erin L. Kuether ◽  
Jocelyn A. Schroeder ◽  
Crystal L. Perry ◽  
Scot A. Fahs ◽  
...  
Keyword(s):  
Protective Effect ◽  
Hemophilia A ◽  
Inhibitory Antibody ◽  
Chromogenic Assay ◽  
Fviii Activity ◽  
Inhibitory Antibodies ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Abstract 709 The important association between von Willebrand factor (VWF) and factor VIII (FVIII) has been investigated for decades, but the effect of VWF on FVIII inhibitors is still controversial. Studies have demonstrated that some anti-FVIII inhibitory antibodies inhibit VWF-FVIII interaction, while others rely on the presence of VWF to inhibit FVIII activities. The influence of VWF on the Bethesda assay, which is routinely used in the clinic to determine the titer of FVIII-neutralizing inhibitors, is still uncertain because the plasma from hemophilia A patients with inhibitors contains normal levels of VWF. To explore the effect of VWF on the reactivity of FVIII inhibitors, we immunized VWF and FVIII double knockout (VWFnullFVIIInull) mice with recombinant human B-domain deleted FVIII (rhFVIII) to induce anti-FVIII inhibitory antibody development. Inhibitory plasma was collected and the titer of inhibitors was determined by Bethesda assay. Murine plasma-derived VWF (from FVIIInull mice) or recombinant human VWF (rhVWF) was used to study the influence of VWF on inhibitor inactivation of FVIII activity (FVIII:C). The remaining FVIII:C after inactivation was determined by chromogenic assay. When inhibitory plasma was incubated with rhFVIII in the presence of 1 U/ml VWF, the residual FVIII activity recovered was higher than in the absence of VWF, resulting in 6.82 ± 1.12 (n = 27) fold lower apparent inhibitor titers. This protective effect is VWF dose dependent. The source of VWF (plasma-derived murine VWF vs. rhVWF) did not affect its protection of FVIII from inhibitor inactivation and VWF does not affect FVIII:C measured in the chromogenic assay in the absence of inhibitors. Interestingly, we found that inhibitor inactivation of FVIII:C in the absence of VWF occurred much faster than in its presence. When the usual 2 hr. incubation at 37°C was omitted from the Bethesda assay, adding rhVWF to rFVIII before mixing with inhibitory plasma resulted in 67.29 ± 20.18 (n = 5) fold lower apparent inhibitor titers than without added VWF. In contrast, if VWF was added to inhibitory plasma first and then mixed with rhFVIII, the inhibitor titers were only 11.04 ± 3.56 (n = 5) fold lower than without added VWF. These results indicate that rhFVIII present in a preformed VWF-FVIII complex is protected from inhibitory antibody inactivation. Conversely, when VWF and inhibitory plasma are added to rhFVIII at the same time, the VWF and inhibitors appear to compete to bind to rhFVIII. Inhibitor titers were lower than in the absence of VWF, but the protective effect is not as efficient as when VWF and rhFVIII were already associated with one another before encountering inhibitors. To confirm the protective effect of VWF on FVIII from inhibitor inactivation, we infused FVIIInull or VWFnullFVIIInull mice with inhibitory plasma and rhFVIII followed by a tail clip survival test. When rhFVIII was infused into FVIIInull mice to 2% followed by inhibitory plasma infusion, all mice with inhibitor titer of 2.5 BU/ml (n = 4) survived tail clipping, and 2 of 4 survived with either 25 BU/ml or 250 BU/ml. If inhibitory plasma was infused first followed by rhFVIII infusion, then only 2 of 6 mice with inhibitor titers of 2.5 BU/ml survived tail clip challenge and none survived with 25 BU/ml and 250 BU/ml. In the first set of mice the infused FVIII was able to form a protective complex with endogenous VWF before encountering inhibitors, while in the second set FVIII is exposed to VWF and pre-infused inhibitory antibodies at the same time, a competitive binding that appears to reduce VWF's protective effect. In contrast, when rhFVIII was infused into VWFnullFVIIInull mice followed by inhibitory plasma infusion, no animals (n = 4 for each group) survived tail clipping with inhibitor titers of 2.5 BU/ml or higher. In summary, our studies demonstrate that VWF exerts a protective effect, reducing inhibitor inactivation of FVIII, both in vitro and in vivo. While the role of VWF in stabilizing plasma FVIII in a milieu rich in proteases has been appreciated for decades, our results indicate that treatment utilizing products containing a complex of FVIII with VWF may be especially beneficial in hemophilia A patients with inhibitors. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Activated platelets induce Weibel-Palade–body secretion and leukocyte rolling in vivo: role of P-selectin

Blood ◽  
2005 ◽  
Vol 106 (7) ◽  
pp. 2334-2339 ◽  
Author(s):  
Vandana S. Dole ◽  
Wolfgang Bergmeier ◽  
Heather A. Mitchell ◽  
Sarah C. Eichenberger ◽  
Denisa D. Wagner
Keyword(s):  
Acute Inflammation ◽  
Endothelial Activation ◽  
Leukocyte Rolling ◽  
Activated Platelets ◽  
Baseline Levels ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Body Secretion

AbstractThe presence of activated platelets and platelet-leukocyte aggregates in the circulation accompanies major surgical procedures and occurs in several chronic diseases. Recent findings that activated platelets contribute to the inflammatory disease atherosclerosis made us address the question whether activated platelets stimulate normal healthy endothelium. Infusion of activated platelets into young mice led to the formation of transient platelet-leukocyte aggregates and resulted in a several-fold systemic increase in leukocyte rolling 2 to 4 hours after infusion. Rolling returned to baseline levels 7 hours after infusion. Infusion of activated P-selectin-/- platelets did not induce leukocyte rolling, indicating that platelet P-selectin was involved in the endothelial activation. The endothelial activation did not require platelet CD40L. Leukocyte rolling was mediated solely by the interaction of endothelial P-selectin and leukocyte P-selectin glycoprotein ligand 1 (PSGL-1). Endothelial P-selectin is stored with von Willebrand factor (VWF) in Weibel-Palade bodies. The release of Weibel-Palade bodies on infusion of activated platelets was indicated by both elevation of plasma VWF levels and by an increase in the in vivo staining of endothelial P-selectin. We conclude that the presence of activated platelets in circulation promotes acute inflammation by stimulating secretion of Weibel-Palade bodies and P-selectin–mediated leukocyte rolling.

scholarly journals Localization of platelet osteonectin at the internal face of the alpha- granule membranes in platelets and megakaryocytes

Blood ◽  
1992 ◽  
Vol 79 (4) ◽  
pp. 936-941 ◽  
Author(s):  
J Breton-Gorius ◽  
P Clezardin ◽  
J Guichard ◽  
N Debili ◽  
L Malaval ◽  
...  
Keyword(s):  
Immunogold Labeling ◽  
Aggregation Process ◽  
Activated Platelets ◽  
Granule Membrane ◽  
Double Label ◽  
Gray Platelet Syndrome ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Fine Localization ◽  
Platelet Content

Abstract Osteonectin is a 32-Kd phosphoglycoprotein originally described in bone but also found in platelets. Platelet and bone osteonectin are different both structurally and immunologically. We have previously shown that platelet osteonectin, by binding to thrombospondin, is involved in the secretion-dependent phase of the platelet aggregation process. In this study, we used antiosteonectin antibodies in combination with immunogold labeling to investigate by electron microscopy the fine localization of osteonectin within normal and gray platelets. Using both a polyclonal and monoclonal antibody ON3, osteonectin was specifically located at the internal face of alpha- granule membranes within normal platelets. Osteonectin was not distributed within all alpha-granules, probably because of its low platelet content. In addition, using immunofluorescence, osteonectin could also be detected in immature and mature megakaryocytes with a granular pattern of staining, suggesting that osteonectin is synthesized by megakaryocytes. Using platelets from two patients with gray platelet syndrome, osteonectin was absent within all abnormal small alpha-granules, but was detected in some rare normal-sized alpha- granules. In separate double-label studies, thrombospondin and von Willebrand factor did not colocalize with osteonectin in resting platelets. However, osteonectin was located at the inner face of the alpha-granules, as it is for alpha-granule membrane protein GMP-140 and glycoprotein IIb-IIIa. These results, taken together with the fact that monoclonal antibodies to osteonectin bind only to the surface of activated platelets, suggest that platelet osteonectin is redistributed to the cell surface during fusion of alpha-granule membranes with the plasma membrane.

scholarly journals von Willebrand factor self-association is regulated by the shear-dependent unfolding of the A2 domain

2019 ◽  
Vol 3 (7) ◽  
pp. 957-968 ◽  
Author(s):  
Changjie Zhang ◽  
Anju Kelkar ◽  
Sriram Neelamegham
Keyword(s):  
Von Willebrand Factor ◽  
Ex Vivo ◽  
Association Studies ◽  
Apolipoprotein A1 ◽  
Platelet Glycoprotein ◽  
Functional Domain ◽  
Self Association ◽  
Von Willebrand ◽  
A2 Domain ◽  
Willebrand Factor

Abstract von Willebrand factor (VWF) self-association results in the homotypic binding of VWF upon exposure to fluid shear. The molecular mechanism of this process is not established. In this study, we demonstrate that the shear-dependent unfolding of the VWF A2 domain in the multimeric protein is a major regulator of protein self-association. This mechanism controls self-association on the platelet glycoprotein Ibα receptor, on collagen substrates, and during thrombus growth ex vivo. In support of this, A2-domain mutations that prevent domain unfolding due to disulfide bridging of N- and C-terminal residues (“Lock-VWF”) reduce self-association and platelet activation under various experimental conditions. In contrast, reducing assay calcium concentrations, and 2 mutations that destabilize VWF-A2 conformation by preventing coordination with calcium (D1498A and R1597W VWD type 2A mutation), enhance self-association. Studies using a panel of recombinant proteins that lack the A1 domain (“ΔA1 proteins”) suggest that besides pure homotypic A2 interactions, VWF-A2 may also engage other protein domains to control self-association. Addition of purified high-density lipoprotein and apolipoprotein-A1 partially blocked VWF self-association. Overall, similar conditions facilitate VWF self-association and ADAMTS13-mediated proteolysis, with low calcium and A2 disease mutations enhancing both processes, and locking-A2 blocking them simultaneously. Thus, VWF appears to have evolved 2 balancing molecular functions in a single A2 functional domain to dynamically regulate protein size in circulation: ADAMTS13-mediated proteolysis and VWF self-association. Modulating self-association rates by targeting VWF-A2 may provide novel methods to regulate the rates of thrombosis and hemostasis.

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