10.4081/btvb.2022.11
Von Willebrand factor (VWF), a blood multimeric protein with a very high molecular weight, plays a crucial role in the primary hemostasis, the physiological process characterized by the adhesion of blood platelets to the injured vessel wall. Hydrodynamic forces are responsible for the VWF multimers conformational transitions from a globular to a stretched linear conformation. These characteristics render this protein a valuable object to be investigated by mechanochemistry, the biophysical chemistry branch that studies the effects of shear forces on protein conformation. This review will focus on the structural elements of the VWF molecule involved in the biochemical response to shear forces. The stretched VWF conformation favors the interaction with the platelet GpIb and at the same time with ADAMTS-13, the zinc-protease that cleaves VWF in the A2 domain, limiting its prothrombotic capacity. It is important to consider the level or the function of VWF or ADAMTS-13 always in relation each other, keeping in mind that in many thrombotic forms of microangiopathies the reduction of the ratio between the ADAMTS-13 activity and the VWF level (lower than 0.5) can be a valuable parameter to predict a real thrombotic risk. Hence, a significant increase in VWF level alone, even without any reduction of ADAMTS-13 concentration, would still be responsible for a significant reduction of the ADAMTS-13/VWF ratio, which ultimately could reflect or predict a prothrombotic risk. Future studies will have to validate the concept whether ADAMTS-13/VWF ratio could a valuable and reliable biomarker to predict or confirm the presence of thrombotic risk in several morbid conditions.
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Raimondo  De Cristofaro
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Bleeding, Thrombosis and Vascular Biology
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The Von Willebrand factor-ADAMTS-13 axis: a two-faced Janus in bleeding and thrombosis
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Bleed Thromb Vascul Biol
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Seth R, McKinnon TAJ, Zhang XF. Contribution of the von Willebrand factor/ADAMTS13 imbalance to COVID-19 coagulopathy. Am J Physiol Heart Circ Physiol 2022;322:H87-H93.
The Von Willebrand factor-ADAMTS-13 axis: a two-faced Janus in bleeding and thrombosis
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                                                                                                                          REVIEW

The Von Willebrand factor-ADAMTS-13 axis: a two-faced Janus in bleeding
and thrombosis
Stefano Lancellotti,1 Monica Sacco,2 Maira Tardugno,2 Antonietta Ferretti,2 Raimondo De Cristofaro1,2
1
  Hemorrhagic and Thrombotic Diseases Service, Fondazione “A. Gemelli” IRCCS University Polyclinic, Rome, Italy; 2Department
of Translational Medicine and Surgery, Faculty of Medicine “A. Gemelli”, Università Cattolica S. Cuore, Rome, Italy



ABSTRACT
     Von Willebrand factor (VWF), a blood multimeric protein with a very high molecular weight, plays a crucial role in the primary he-
mostasis, the physiological process characterized by the adhesion of blood platelets to the injured vessel wall. Hydrodynamic forces
are responsible for the VWF multimers conformational transitions from a globular to a stretched linear conformation. These character-
istics render this protein a valuable object to be investigated by mechanochemistry, the biophysical chemistry branch that studies the
effects of shear forces on protein conformation. This review will focus on the structural elements of the VWF molecule involved in the
biochemical response to shear forces. The stretched VWF conformation favors the interaction with the platelet GpIb and at the same
time with ADAMTS-13, the zinc-protease that cleaves VWF in the A2 domain, limiting its prothrombotic capacity. It is important to
consider the level or the function of VWF or ADAMTS-13 always in relation each other, keeping in mind that in many thrombotic




                                                                                            ly
forms of microangiopathies the reduction of the ratio between the ADAMTS-13 activity and the VWF level (lower than 0.5) can be a
valuable parameter to predict a real thrombotic risk. Hence, a significant increase in VWF level alone, even without any reduction of




                                                                                     on
ADAMTS-13 concentration, would still be responsible for a significant reduction of the ADAMTS-13/VWF ratio, which ultimately
could reflect or predict a prothrombotic risk. Future studies will have to validate the concept whether ADAMTS-13/VWF ratio could a
valuable and reliable biomarker to predict or confirm the presence of thrombotic risk in several morbid conditions.



                                                                                e
                                                                           us
                                                                            dothelial injury. This process is mostly mediated by Von
THE MAIN PLAYERS OF THE GAME:                                               Willebrand factor (VWF), a multimeric protein of variable
VON WILLEBRAND FACTOR AND ADAMTS-13
                                                                           al
                                                                            molecular weight ranging from about 0.5 to about 20
   Vascular rupture is rapidly counteracted by adhesion                     megadalton, which acts as a bridge between the suben-
                                                        ci


and firm attachment of blood platelets at the site of en-                   dothelial collagen and platelets.1,2 VWF multimers con-
                                                      er



                                                                            nect the platelet receptor GpIb/IX/V to collagen fibrils of
                                                                            the extracellular matrix.2,3 VWF immobilized on suben-
                                                     m




  Correspondence: Raimondo De Cristofaro, Dipartimento di Med-
                                                                            dothelial collagen forms in fact a reactive surface, which
                                          om




  icina e Chirurgia Traslazionale, Facoltà di Medicina e Chirurgia          captures platelets from flowing blood.4 Notably, at high
  “A. Gemelli”, Università Cattolica S. Cuore, Roma; Fondazione             shear stress (>30 dyn/cm2) VWF undergoes micro- and
  Policlinico Universitario “A. Gemelli” IRCCS Servizio Malattie
  Emorragiche e Trombotiche, Largo F. Vito 1, 00168 Rome, Italy.
                                                                            macro-conformational changes from a globular state to a
                                   -c




  Tel.: +39-0630156329 - Fax: +39-06-30155915.                              stretched conformation, where especially the A2 domain
  E-mail: raimondo.decristofaro@unicatt.it                                  undergoes relevant structural changes.5-7 VWF mediates
                          on




  Key words: Von Willebrand Factor, Shear stress, Mechanochem-              platelet adhesion to the damaged vessel wall under con-
  istry, Hemostasis, Platelets.                                             ditions of high shear stress. The VWF gene, localized on
                    N




                                                                            chromosome 12, spans 178 kb and contains 52 exons. It
  Contributions: RDC wrote the initial draft of the manuscript. SL,
  MS, MT and AF critically reviewed the draft. All authors read and         is transcribed into an 8.8-kb messenger RNA, which en-
  commented on the paper and approved submission.                           codes a 2813-aa precursor (pre-pro-VWF) consisting of a
  Conflict of interest: The author declares no potential conflict of in-
                                                                            22-aa signal peptide, a 741-aa propeptide and a 2050-aa
  terest.                                                                   mature subunit.1,8,9 The VWF mature subunit is composed
                                                                            of 4 repetitive domains designated A to D and arranged
  Funding: RDC gratefully acknowledges financial support from the
  Catholic University (“Linea D1-AA 2019).                                  in the sequence D’-D3-A1-A2-A3-D4-C1-C2-C3-C4-C5-
                                                                            C6-CK.5.1 The platelet GpIbα receptor binding to the A1
  Received for publication: 1 January 2022.                                 domain of immobilized VWF results in an initial adhe-
  Accepted for publication: 1 April 2022.
                                                                            sion, and a continuous surface translocation of the
  This work is licensed under a Creative Commons Attribution Non-           platelets.10,11 This process ends with a stable platelet ad-
  Commercial 4.0 License (CC BY-NC 4.0).                                    hesion through interaction with the platelet collagen re-
  ©
   Copyright: the Author(s), 2022                                           ceptors GpVI and α2β1 integrin (GpIa/IIa),12-14 activation
  Licensee PAGEPress, Italy                                                 of the platelet GpIIb/IIIa receptor complex and finally
  Bleeding, Thrombosis and Vascular Biology 2022; 1:11
  doi:10.4081/btvb.2022.11
                                                                            platelet-platelet aggregation. During the process of pri-
                                                                            mary hemostasis, the interaction between VWF and

                                              Bleeding, Thrombosis and Vascular Biology 2022; 1:11
10                                                     S. Lancellotti et al.




GpIbα does not occur under static conditions, but it needs           contains 19 members that share the common structure of
a pre-activation of soluble VWF. As anticipated, this                a hydrophobic signal sequence, a propeptide, a metallo-
process consists of a conformational change of VWF mol-              protease domain (M), a thrombospondin type 1 (TSP1)
ecules, which expose within the A1 domain the binding                repeat, a disintegrin-like domain (Dis), a cysteine-rich do-
site for GpIbα. This mechanism is triggered either by me-            main (Cys-R), and a spacer domain (Spa) (Figure 1A).38
chanical forces, such as a high shear rate (>5000 s-1), or           In addition, all ADAMTS family members have one or
shear stress >30 dyn/cm2, present in the microcirculation.           more thrombospondin type 1 (TSP1) motifs and variable
The same effect can also be obtained by chemical poten-              additional C-terminal domains. The carboxyl terminus of
tials generated by the interaction with biochemical effec-           ADAMTS-13 contains seven more TSP1 repeats and two
tors, such as the antibiotic glycopeptide ristocetin, the            CUB domains, which are named after motifs first identi-
snake venom botrocetin and polyphosphate.5,6,15-21 How-              fied in Complement components C1r and C1s, sea urchin
ever, even natural mutations causing some types 2B von               protein uEGF, and Bone morphogenetic protein-1, which
Willebrand disease (VWD), such as the Arg1306Trp,                    can interact with the D4 VWF domain even under static
Arg1341Trp,             His1268Asp,            Arg1306Gln,           conditions.39 A recent X-ray diffraction study showed that
Arg1308Cys/Leu, Ile309Val, Val1316His, Pro1337Leu,                   the ADAMTS-13 CUB1-2 domains have two highly con-
and Arg1341Gln/Trp or in the A1 domain,22-24 stabilize a             served surface patches that form an extended binding site
stretched conformational state that favors the interaction           for the central ADAMTS-13 Spacer domain (Figure 1B).40




                                                                                              ly
with the GpIb platelet receptor even under very low shear            The interaction of the CUB1-2 domains with the Spacer




                                                                                       on
conditions.25 However, not all the types 2B VWD variants             domain regulates the ADAMTS-13 global latency by
exhibit the same enhanced interaction with platelets, re-            locking ADAMTS-13 in a closed conformational status,
vealed by the inverse correlation with platelet count.24             which is only released, physiologically, when ADAMTS-



                                                                                 e
This may derive from the fact that specific amino acid               13 interacts with VWF.40,41 Thus, not only VWF under-
substitutions are probably critical for differently influenc-
                                                                           us
                                                                     goes extensive conformational transition under the effect
                                                                     of high shear stress, but even ADAMTS-13 is subjected
ing the conformational equilibria and thus type 2B VWD
phenotypes. The stretched conformation of VWF not only               to allosteric transitions upon its interaction with VWF, re-
                                                                    al

allows the interaction with the platelet GpIb receptor, but          sembling a sort of sequential model of allosteric
                                                                     behavior.42 Experimental steady-state enzymatic studies,
                                                      ci

also the proteolytic attack by the zinc-protease ADAMTS-
13 (A Disintegrin And Metalloproteinase with a Throm-                which showed a hyperbolic mixed-type inhibition of the
                                                    er



bospondin type 1 motif, member 13).7,25-29 which, cleaving           hydrolysis by ADAMTS-13 of a VWF peptide sub-
                                                                     strate,28,29 also confirmed this model.43,44 In this review, the
                                                   m




the Tyr1605-Met1606 in the A2 domain of VWF, prote-
olyzes the molecule and limits the hemostatic and even               relationship between the ADAMTS-13 and VWF levels
                                          om




prothrombotic activity of the ultra large VWF multimers.7            (ADAMTS-13/VWF ratio) will be discussed concerning
A shear stress >30 dyn/cm2 confers the ability to VWF                its potential role as a biomarker of thrombotic and vascu-
multimers to interact with the platelet GpIb receptor and            lar disorders.
                                     -c




at the same time to be fragmented by ADAMTS-13 at the
Y1605-M1606 peptide bond in the A2 domain. Further-
                             on




more, a high shear force favors a VWF self-association, a            MECHANISMS OF SHEAR-INDUCED
process by which multimeric VWF binds to or aggregates               REGULATION OF THE ADAMTS-13/VWF
                         N




with additional VWF multimers. This process is respon-               INTERACTION
sible for the formation of VWF fibers, that are organized                The high sensitivity of VWF to hydrodynamic forces
as spider-web like structures.5,30 This network constitutes          raises the question concerning the molecular mechanisms
an ideal substrate for recruiting flowing platelets in the           by which VWF multimers undergo major conformational
circulation during the process of primary hemostasis. All            changes responsible for the acquisition of their full pro-
these VWF properties stem from the unique capacity of                hemostatic and even prothrombotic activities. The inter-
this multimeric protein to be very sensitive to external hy-         action of VWF multimers with cell receptors and other
drodynamic forces as many different biological                       proteins were investigated by using the principles of
processes,5 such as cell adhesion, cytoskeleton organiza-            mechanochemistry.45 The principles of mechanochemistry
tion, cell division, bacteria invasion, and the activity of          find a proper application in biological systems, where
ion channels.31-37                                                   macromolecules in living systems do not often interact
    The enzyme responsible of the VWF proteolysis is                 each other freely in solution, according to ideal kinetics
ADAMTS-13. This enzyme is the 13th member of the                     and thermodynamics principles, but frequently interact
ADAMTS family of zinc proteases, which is related to                 tethered to other subcellular or matrix components and in
the large ADAM (A Disintegrin and Metalloprotease)                   a highly structured mode. To characterize the acting bio-
family. The ADAMTS family of zinc metalloproteases                   physical mechanisms, different types of bonds were iden-

                                     Bleeding, Thrombosis and Vascular Biology 2022; 1:11
                   The Von Willebrand factor-ADAMTS-13 axis: a two-faced Janus in bleeding and thrombosis                   11



tified, such as the catch-slip bond,46-48 and flex bond.49        arity of VWF interaction with cell receptors or other pro-
These binding models assume that the interaction between          teins resides in its biochemical plasticity, which allows
two macromolecules depends on external forces that ex-            the protein to follow ideal or mechanochemical pathways
ponentially accelerate the dissociation between interacting       in performing its biological functions, as described below.
particles or ideal bond,50,51 indicating that the bond life-
time is independent of an externally applied force. No-
tably, the flex bond was demonstrated to occur in the
                                                                  THE EFFECT OF SHEAR FORCES ON
binding of the VWF A1 domain to GpIb.49 By using the
                                                                  VON WILLEBRAND FACTOR IN DISEASE
ReaLiSM method (repeated measurements of the binding
                                                                  AND NORMAL CONDITIONS
and unbinding of a receptor and ligand in a single mole-
cule), Kim et al. showed the existence of two states of the           Although most of the interactions that VWF can make
receptor-ligand bond, that is, a flex-bond.49 One state was       with many macromolecular ligands are possible only if
observed at low hydrodynamic force (<5 pN), whereas a             VWF assumes a stretched conformation under high shear
second state begins to engage at about 10 pN with a 20-           forces, the interaction with subendothelial collagen or other
fold longer lifetime and higher force resistance. The sec-        molecules can take place even by globular VWF con-
ond state can resist to flowing forces that could                 former, as collagen was demonstrated to be rather insensi-
disaggregate the platelet plug. Although many experimen-          tive to hydrodynamic forces.52-54 Collagen III plays the most




                                                                                   ly
tal evidences were provided by different authors, no de-          relevant role in the interaction with the VWF A3 domain,




                                                                            on
finitive conclusion has been reached on what type of bond         whereas type VI collagen has only an ancillary role, useful
stabilizes and/or controls VWF interaction with platelets         in the case of natural mutations that impair the A1 interac-
or the vessel wall.45 It must be remarked that the peculi-        tion with type III collagen.52 As anticipated above, the in-



                                                                       e
                                                                us
                                                          al
                                              ci
                                            er
                                           m
                                  om
                            -c
                     on
                 N




Figure 1. A) Cartoon showing the global structure of the ADAMTS-13 structure. B) Crystal structure of the CUB1-2 conjugated to
maltose-binding protein (MBP) (PDB code «7B01») (40). The image rendering was performed with the PyMOL program.


                                     Bleeding, Thrombosis and Vascular Biology 2022; 1:11
12                                                       S. Lancellotti et al.




teraction between the VWF A1 domain and the GpIb/IX/V                  that, occurring mostly in the A2 domain, propagate there-
platelet receptor can occur only in the microcirculation,              after through the A1 and other domains of the monomer,
where a shear rate >2000 sec-1 is generated. From the                  causing ultimately a stretching of the multimers.
mechanochemistry standpoint, the interaction between
VWF and collagen can be characterized by an “ideal bond”,
independently from external applied forces. This finding               THE A2 DOMAIN OF VWF IS A
agrees with the possibility to measure the interaction be-             MECHANOSENSITIVE SWITCH
tween HMW-VWF with collagen in ELISA tests even
                                                                           The X-ray crystal structure of the A2 domain showed
under static conditions in the common diagnostic practice
                                                                       that it resembles the overall conformation of the so-called
of von Willebrand disease (VWD).55 Another type of ideal
                                                                       VWA domain,6 present in a protein superfamily comprising
bond interaction concerns the VWF binding to the low-den-
                                                                       integrins on cell surfaces, complement components and
sity lipoprotein receptor-related protein-4 (LRP4), a newly
                                                                       DNA repair proteins.61 However, the A2 domain shows a
identified receptor for VWF on smooth muscle cell of ves-
                                                                       unique structure if compared to other VWA domains. In
sel wall.56 This interaction seems to play a central role in
                                                                       fact, the A2 domain has a sequence of α-helices and β-
the vessel intima hyperplasia, favouring ischemic diseases.
                                                                       strands that alternate in sequence (Figure 2). The hydrody-
VWF multimers bound to subendothelial collagen form a
                                                                       namic shear forces act mostly on this domain, as the
reactive surface capable of capturing platelets from circu-
                                                                       unfolding will proceed from the C-terminal end rather than




                                                                                                 ly
lating blood.57,58 Furthermore, when VWF multimers are
                                                                       the N-terminal end of the A2 domain, because the C-termi-




                                                                                         on
bound to collagen, their rotational entropy, present when
                                                                       nal structural elements can be more easily moved one at a
the protein is free in solution, is drastically reduced and the
                                                                       time from the end of the domain, while the β1 strand at the
elongational force becomes predominant.
                                                                       N-terminal is surrounded by counterforts constituted by β2


                                                                                    e
                                                                       and β4 strands in the core of the domain (Figure 2). Based
                                                                             us
                                                                       on this structural arrangement, any external hydrodynamic
THE ROLE OF SHEAR FORCES IN DIFFERENT
                                                                       force acting on the C-terminus of the A2 domain will be si-
FORMS OF VON WILLEBRAND DISEASE
                                                                       multaneously sensed by the 8-membered Cys1670-
                                                                      al

    The shear force acts on the multimers so that the latter           Cys1669 disulfide ring, which acts a high energy barrier to
                                                        ci

form an elongated strain on the endothelial surface that
exposes multiple binding sites for ligands and receptor.
                                                      er



Finally, even gain-of-function natural mutations causing
                                                     m




type 2B VWD, such as the p.R1306W or p.R1341W, are
able to stabilize a conformational state that is prone to the
                                           om




interaction with the platelet receptor GpIb even under
very low shear conditions, thus causing platelet clumping
in the systemic circulation.23,59 As anticipated above, a
                                      -c




shear stress level >30 dyn cm2 would cause the unfurling
                              on




of VWF multimeric strings. This stretching phenomenon
can expose the A1 domain that binds to the platelet GpIb.
However, under certain shear stress conditions the macro-
                         N




conformational transition is accompanied by a degree of
molecular flexibility that allows the VWF A1 residues en-
gaged in the binding to GpIb to fit into the interacting cleft
of the receptor.60 Natural mutations causing type 2M
VWD, such as p.G1324A and p.G1324S, generate a more
rigid sequence due to the substitution of the simple side
chain of glycine (hydrogen atom) with a larger side chain
(methyl group and hydroxyl group of alanine and serine,
                                                                       Figure 2. Ribbon diagram of VWF A2 domain of human A2 do-
respectively) that strongly limits the conformational flex-            main (PDB code “3GXB”) (6). The red cross represents the hy-
ibility of that part of the molecule contained in the β-turn           dration water molecule. Note the presence of several helices at
between β-strands 2 and 3 within the native state of                   the lateral sides of the molecule and the b-strands at the mid of
VWF.60 Hence, not even external hydrodynamic forces                    the molecule. The shear forces push from the C-terminus toward
are able to counteract the rigidity of this segment of the             the N-terminal of the molecule to induce the stretching of the
protein, which has become much more rigid due to the                   domain with the exposition of the Tyr1605-Met1606 peptide
type 2M mutations. By contrast, in the wild type VWF                   bond cleaved by ADAMTS-13. The image rendering was per-
hydrodynamic forces induce conformational transitions                  formed with the PyMOL program.


                                       Bleeding, Thrombosis and Vascular Biology 2022; 1:11
                    The Von Willebrand factor-ADAMTS-13 axis: a two-faced Janus in bleeding and thrombosis                     13



the external force. Once this energetic barrier is overcome,       many binding sites available for interactions with cell re-
the external force will be transmitted to the hydrophobic          ceptors and other protein ligands.
core containing the Tyr1605-Met1606, because of the apo-
lar interactions with Leu1603 and Tyr1605. At this point,
the poor packing of the β4 strand and the high conforma-           THE ROLE OF THE MOLECULAR DIMENSION
tional flexibility of the long loop replacing the α4 strand        OF VWF MULTIMERS IN SHEAR-INDUCED
give rise to an easy unfolding of the hydrophobic core. The        STRETCHING
unfolding of the inner hydrophobic core of the A2 domain
                                                                       The unfolding of A2 domains, occurring under a ≈11
is accompanied by solvation of the region and the Tyr1605-
                                                                   pN tensile force,6 and its hydrolysis of by ADAMTS-13,
Met1606 becomes available to the proteolysis by
                                                                   occurs preferentially near the center of untangled multi-
ADAMTS-13. The structural peculiarity of the vicinal
                                                                   mers. The ultra large VWF multimers undergo rapid scis-
disulfide Cys1669-Cys1670 plays a central role in the
                                                                   sion upon secretion into the vasculature because of their
mechanosensitive properties of the entire A2 domain,
                                                                   length.63 In fact, the tensile force F(j) to the inside of any
whose genetic mutations are largely responsible for many
                                                                   sphere pair j in a chain with N dimers, is the sum of the
forms of type 2A von Willebrand disease (p.R1597W,
                                                                   force on all the outer dimer pairs. Hence, the total tensile
p.G1505E, p.I1628T, and p.E1638K) that shows a loss of
                                                                   force is given by7,64
high molecular weight multimer because of an increased




                                                                                    ly
proteolysis by ADAMTS-13 in the bloodstream. The su-




                                                                             on
pernormal proteolysis by ADAMTS-13 is linked to the ten-
dency of these VWF mutants to be in a more “open”,
unfolded conformations that render the Y1605-M1605 in                  Where f(i) is the normal force between two spheres that



                                                                        e
the hydrophobic core of the β4 strand susceptible to the pro-      are a certain distance (x) apart, d is the length of the rigid
teolytic attack by the metalloprotease. The activity of high
                                                                 ustether between two monomers, and a is the radius of the
                                                                   monomer. From eq. 1, the normal force on a monomer in
shear forces triggers a shift toward the unfolded form of the
A2 domain, as unraveled by the X-ray diffraction studies           the center of a multimer is roughly proportional to N2 (i.e.,
                                                            al

by Zhang et al.6 It must be outlined that in the flowing           when j=1), whereas the force on a monomer at the end of a
                                               ci

blood VWF multimers are exposed to high hydrodynamic               multimer is proportional to N (i.e., when j = N). This quad-
forces for a limited time and, due to the reversibility of the     ratic dependence markedly separates longer and shorter
                                             er



unfolding reaction, ADAMTS-13 could exert its proteolytic          multimers, if referred to the capacity of external shear force
                                                                   to stretch the molecule. Zhang et al. calculated that 11 pN
                                            m




activity for a very short time.28,29 However, the nature has
provided VWF with a biochemical mechanism that allows              can unfold an upper length limit for VWF of 200
                                   om




conversion from a cis- to a trans W1644-P1645 peptide              monomers, as observed in atomic force experiments.7 Thus,
bond.62 This structural transition is capable to greatly delay     it should be outlined that the length of VWF multimers is
the process of refolding of the A2 domain, thus allowing a         essential for their hemostatic function: short multimers are
                             -c




more prolonged activity by ADAMTS-13. Once the unfold-             not able to reach sufficient forces to induce the conforma-
ing of the A2 domain takes place, conformational transi-           tional transition described above. Another advantage of
                      on




tions involve also the vicinal A1 and A3 domains, so that          having sufficiently long multimers resides in their ability
the binding sites for other ligands become available for in-       to expose more interaction sites than shorter chains. In other
                 N




teractions. Hence, the conformational instability of the A2        words, the stretching mechanism can increase the stoi-
domain under sufficient shear forces can also affect the in-       chiometry of VWF/ligand interaction, comprising
teraction between the A1 and A2 domains. These two do-             ADAMTS-13. The occurrence of these biophysical mech-
mains are adjacent to each other and connected by a linker         anisms finds its expression in pathological conditions re-
of 30 amino acids. Under low shear conditions, the A1 do-          sponsible for opposite effects. Inherited VWF diseases
main, and particularly the GpIb binding site, is shielded by       characterized by severe deficiency of high molecular
a specific inhibitory interaction with the vicinal A2              weight VWF multimers (e.g. type 2A VWD) cause hemor-
domain.53 Thus, once enough shear force overcomes the re-          rhages, whereas in the acquired disease referred to as
sistance of the A2 domain to unfold, the conformational            thrombotic thrombocytopenic purpura, the accumulation of
transition in the A2 domain gives rise to two different ef-        ultra-large VWF multimers, due to deficiency of
fects: 1) the exposition of the Y1605-M1606 to solvent,            ADAMTS-13, causes thrombotic complications in the mi-
rendering it available to the proteolytic attack by                crocirculation, where high shear stress is generated.65-67 Nat-
ADAMTS-13; 2) the elimination of the shielding effect of           ural mutations of the VWF A1 domain may differently
the A2 on the A1 domain with exposition of the GpIb bind-          affect the conformational transitions of the whole VWF
ing site in the A1 domain. Furthermore, the shear force-de-        multimer linked to external hydrodynamic forces, as no-
pendent stretching of the whole VWF multimer creates               ticed above for the type 2M von Willebrand mutations

                                      Bleeding, Thrombosis and Vascular Biology 2022; 1:11
14                                                     S. Lancellotti et al.




p.G1324A and p.G1324S, as described above. This effect               Michaelis-Menten approximations considering steady
is associated with resistance to unfolding induced by dena-          state conditions, being the concentration of the substrate
turing agents, reduced sensitivity to hydrodynamic force             VWF>ADAMTS-13, as found in blood in physiological
and decrease of affinity for GpIb under shear flow and re-           conditions (ADAMTS-13: 3-4 nM; VWF:Ag: ≈40 nM).
sistance to limited proteolysis.60 By contrast, the natural          However, as anticipated in the preceding paragraph the
mutation R1306W in the A1 domain, causing a type 2B von              interaction between ADAMTS-13 and VWF shows a par-
Willebrand disease, switches the equilibrium toward an un-           ticular cooperativity, being characterized by a hyperbolic
folded conformation of VWF multimers, thus showing a                 mixed-type inhibition by product.28,29 Hence, the higher
higher sensitivity to shear stress,23 which facilitates expo-        the ultra-large VWF concentration the higher is the inhi-
sure of GPIb binding sites. The mean hydrodynamic diam-              bition mechanism by its substrate (Figure 3). Under cer-
eter of resting p.R1306W VWF multimers was indeed                    tain clinical conditions (inflammatory status, cancer,
significantly greater than that of wild type VWF multimers           cardiovascular diseases, viral infections, etc.) where a
(210±60 nm vs. 87±22 nm, respectively).60 At shear forces            massive secretion of VWF may occur, the excess of the
<14 dyn cm2, the p.R1306W multimers rapidly populates                substrate causes a functional insufficiency of ADAMTS-
stretched conformers, acquiring the capacity to interact with        13 to proteolyze ultra-large VWF with a progressive ac-
GpIb which, instead, was induced for WT VWF by shear                 cumulation of these multimers. These conditions are
forces >30 dyn cm2 only.60                                           characterized by a marked decrease of the ADAMTS-




                                                                                               ly
     Another relevant VWF activity gained upon attainment            13/VWF ratio <0.5 can be considered a biomarker of en-




                                                                                       on
of a stretched conformation under appropriate shear forces           hanced risk of thrombotic disorders.72
is the propensity to self-aggregate.5 Thus, when the unfold-
ing prevails under the effect of shear forces, VWF multi-



                                                                                 e
mers act as promoters of thrombotic mechanisms but at the            THE RELEVANCE OF THE ADAMTS-13/VWF
same time are available to the proteolysis by ADAMTS-
13. When this proteolytic process cannot take place, due to
                                                                           us
                                                                     RATIO IN ISCHEMIC/THROMBOTIC
                                                                     AND HEMORRHAGIC DISORDERS
the formation of autoantibodies against ADAMTS-13 or
                                                                         The relative levels of both ADAMTS-13 and VWF in
                                                                    al
for genetic deficiency of the metalloprotease, thrombotic
                                                                     healthy subjects can be described by a ratio close to unity
microangiopathy occurs, characterized by an accumulation
                                                      ci

                                                                     (1.0), although it may range from about 0.5 to 2.0. When
of ultra-large VWF multimers.66,68 Upon stabilization of the
                                                    er



stretched conformation, the swapped domains of a VWF
multimer can interact with the same domain but belonging
                                                   m




to a different multimer. Hence, the non-covalent intra-
                                          om




monomer interactions between domains are broken and re-
stored between different multimer chains: this aggregation
mechanism may be an example of the biophysical phenom-
                                     -c




enon referred to as 3D domain swapping.69,70 The latter is a
mechanism that allows protein molecules to form dimers
                             on




or polymers by exchanging identical domains. More prop-
erly, we should consider the process of VWF multimeriza-
                         N




tion and subsequent molecular aggregation as a 3D domain
swapping mechanism.

                                                                     Figure 3. Simulation effect of the hyperbolic mixed-type inhi-
THE ADAMTS-13/VWF CONCENTRATION                                      bition by hydrolyzed VWF (product) on the inhibition degree of
RATIO: A DELICATE BALANCE BETWEEN                                    the hydrolysis of VWF as substrate at the indicated concentra-
HEMOSTASIS AND THROMBOSIS                                            tion. The points were calculated by the appropriate parameters
                                                                     of the hyperbolic mixed-type inhibition equation, as previously
    The kinetics of the interaction between the zinc-pro-            reported: Vi= kcat * ESZ°/[(KmZ1) + (SZ2)], where Z° = 1+
tease ADAMTS-13 and its substrate VWF has been ex-                   (βI/αKi); Z1= 1 + (I/Ki); Z2 = 1 + I/αKi) and Km of VWF cleavage
tensively investigated.28,29,71 Under “physiological”                by ADAMTS-13 equal to 1 µM, kcat=1sec-1, Ki is the equilibrium
conditions of temperature, pH, and calcium/zinc/chloride             dissociation constant of cleaved product binding to ADAMTS-
ion concentration, under steady state conditions                     13= 1 µM, a=10 (the corrective factor for Ki when the product
ADAMTS-13 cleaves VWF at the Tyr1605-Met1606 pep-                    binds to the ES complex), and b=0.23 (the corrective factor for
tide bond with a Km in the low µM range, with a kcat of              kcat concerning the ESI adduct) (18). VWF:Ag level 100%=10
about 1-2 sec-1, and a kcat/Km of ≈5 x105 M-1 sec-1.28,29            µg/ml≈40 nM. The calculations were performed with the Graph-
                                                                     Pad Prism software.
These parameters were obtained using in vitro the

                                     Bleeding, Thrombosis and Vascular Biology 2022; 1:11
                       The Von Willebrand factor-ADAMTS-13 axis: a two-faced Janus in bleeding and thrombosis                                 15



this ratio falls in this range the VWF/ADAMTS-13 axis is
balanced. In VWD, the ADAMTS-13/VWF ratio>>2.0 or                             WHAT ABOUT THE ROLE OF THE
approaching infinity in the case of type 3 VWD. At vari-                      ADAMTS-13/VWF AXIS IN EXTRA-HEMOSTATIC
ance with congenital hemorrhagic disorders, in thrombotic                     FUNCTIONS OF VWF
microangiopathies, such as acquired or congenital severe                      AND THE METALLOPROTEASE?
Thrombotic Thrombocytopenic Purpura (TTP) the                                     It has definitively been established that the biological
ADAMTS-13/VWF ratio approaches zero. The ADAMTS-                              roles of VWF and ADAMTS-13 are not limited to hemo-
13/VWF ratio has also been reported to have clinical sig-                     stasis, as they are also engaged in other processes, such
nificance in several ischemic/thrombotic conditions. For                      as angiogenesis and cell proliferation.85-89 VWF also un-
instance, as a predictor of outcome in acute ischemic brain                   dergoes a secretion through the basal pole of the endothe-
injury,73,74 predicting thrombotic complications in cirrhosis                 lial cells, thus being present also in the subendothelial
and post-hepatectomy,75-77 and as a prognostic factor after                   space. A very recent study has demonstrated that basal se-
acute myocardial infarction or infection by Sars-CoV-2.78-                    cretion by endothelial cells of Weibel-Palade bodies
81
   In these vascular conditions the scenario is dominated by                  causes subendothelial and abluminal VWF retention that
an increase of ultra-large VWF multimers. This occurs                         stimulates smooth muscle cell (SMC) proliferation
under the pressure of inflammatory cytokines or other sec-                    through the activation of low-density lipoprotein recep-
retagogues substances (adrenergic molecule, thrombin,                         tor-related protein-4 (LRP4) in cooperation with integrin




                                                                                                  ly
FXa, etc.) that facilitate the secretion of Weibel-Palade                     aVb3 by the A2 domain of VWF.56 It is still unknown




                                                                                         on
granules containing the ultra-large forms of VWF. If in this                  whether ADAMTS-13 is present in the subendothelial
situation the ADAMTS-13 level remains constant or even                        spaces and whether can proteolytically process VWF in
decreases, the physiological stoichiometry ratio between                      this extravascular milieu. However, the available prelim-


                                                                                   e
ADAMTS-13 and VWF is significantly reduced, favoring                          inary data pave the way to a wider comprehension of
the presence in the circulation of the ultra-large forms of
                                                                           us many biological functions performed by VWF and
VWF. Clinical trials are underway to demonstrate the                          ADAMTS-13. Whether the ADAMTS-13/VWF ratio
pathogenetic role of a reduced ADAMTS-13/VWF ratio in                         plays a significant role even in these extra-hemostatic
                                                                    al
various cardiovascular and thrombotic diseases and its use                    functions of the proteins is not yet discovered, although
                                                     ci

as a prognostic biomarker of the outcome. At variance with
                                                                              we can hypothesize that this is the case.
the above settings, in other clinical conditions, such as those
                                                   er



found in subjects with type O blood group, patients with
some type 2A VWF variants, or bearing ventricular assisted
                                                  m




                                                                              CONCLUSIONS
devices, the interaction between ADAMTS-13 and VWF
                                         om




multimers is facilitated, due to altered VWF conformation.                        In this review, we have described how the high sensitiv-
This effect induces the exposition to solvent of the                          ity of VWF multimers to mechanical forces, which is cen-
Tyr1605-Met1606 peptide bond in the A2 domain that fa-                        tered in the A2 domain, plays a pivotal role in the
                                  -c




vors the proteolytic attack by ADAMTS-13,82-84 causing the                    pathophysiology of several hemorrhagic and thrombotic dis-
loss of high molecular weight VWF multimers and hemor-                        orders. However, we should always keep in mind that when
                             on




rhagic diathesis. Table 1 summarizes the different condi-                     we consider the level or the function of VWF or
tions, in which the perturbation of the ADAMTS-13/VWF                         ADAMTS13 alone, without considering the other partner,
                    N




ratio is associated with thrombotic and hemorrhagic phe-                      this can lead to a limited interpretation of clinical or exper-
notypes.                                                                      imental findings. Hence, a significant increase in VWF level


Table 1. Abnormal ADAMTS-13/VWF ratio in various thrombotic and hemorrhagic disorders.
ADAMTS-13/VWF ratio                Clinical condition                  VWF pattern                        Symptom               References
<<0.50                               Acquired TTP           High molecular weight multimers ↑     Microcirculatory thrombosis    (66, 68)
<0.50                            Acute hepatic failure        High molecular weight multimers ↑          Thrombosis    (73, 75-77, 79-81, 90, 91)
                              with portal vein thrombosis.
                                  Cerebral ischemia.
                             Endothelial dysfunction in viral
                                       infection.
≅1 or >1 but with                  Blood group 0.           High molecular weight multimers ↓            Hemorrhage              (82-84)
enhanced VWF                       Type 2A VWD.
susceptibility to cleavage       VAD-bearing patients.
by ADAMTS-13
VAD, Left Ventricular-Assisted Device.


                                           Bleeding, Thrombosis and Vascular Biology 2022; 1:11
16                                                         S. Lancellotti et al.




alone, even without any reduction of ADAMTS13 concen-                    16. Di Stasio E, Romitelli F, Lancellotti S, et al. Kinetic study
tration, would still be responsible for a significant reduction              of von Willebrand factor self-aggregation induced by risto-
of the ADAMTS-13/VWF ratio and could ultimately reflect                      cetin. Biophys Chem 2009;144:101-7.
                                                                         17. Papi M, Maulucci G, De Spirito M, et al. Ristocetin-induced
or predict a prothrombotic risk. Further studies must be con-
                                                                             Self-Aggregation of Von Willebrand Factor. Eur Biophys J
ducted to validate the concept whether ADAMTS-13/VWF                         2010;39:1597-603.
ratio could a valuable and reliable biomarker to predict or              18. Di Stasio E, De Cristofaro R. The effect of shear stress on
confirm the presence of prothrombotic conditions.                            protein conformation: Physical forces operating on bio-
                                                                             chemical systems: The case of von Willebrand factor. Bio-
                                                                             phys Chem 2010;153:1-8.
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