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Keywords:

  • ADAMTS13 protein;
  • animals;
  • arterial thrombosis;
  • autoimmune diseases;
  • factor VIII;
  • human;
  • thrombotic microangiopathies;
  • von Willebrand factor (445–733)

Summary

  1. Top of page
  2. Summary
  3. ADAMTS-13 and potential human diseases
  4. Biosynthesis and secretion of ADAMTS-13
  5. Structure–function of ADAMTS-13
  6. Regulation of ADAMTS-13 function
  7. Mechanism of autoantibody inhibition
  8. Improving on nature, re-engineering ADAMTS-13
  9. Acknowledgements
  10. Disclosure of Conflict of Interest
  11. References

ADAMTS-13, a plasma reprolysin-like metalloprotease, cleaves von Willebrand factor (VWF). Severe deficiency of plasma ADAMTS-13 activity results in thrombotic thrombocytopenic purpura (TTP), while mild to moderate deficiencies of plasma ADAMTS-13 activity are emerging risk factors for developing myocardial and cerebral infarction, pre-eclampsia, and malignant malaria. Moreover, Adamts13−/− mice develop more severe inflammatory responses, leading to increased ischemia/perfusion injury and formation of atherosclerosis. Structure–function studies demonstrate that the N-terminal portion of ADAMTS-13 (MDTCS) is necessary and sufficient for proteolytic cleavage of VWF under various conditions and attenuation of arterial/venous thrombosis after oxidative injury. The more distal portion of ADAMTS-13 (TSP1 2–8 repeats and CUB domains) may function as a disulfide bond reductase to prevent an elongation of ultra-large VWF strings on activated endothelial cells and inhibit platelet adhesion/aggregation on collagen surface under flow. Remarkably, the proteolytic cleavage of VWF by ADAMTS-13 is accelerated by FVIII and platelets under fluid shear stress. A disruption of the interactions between FVIII (or platelet glycoprotein 1bα) and VWF dramatically impairs ADAMTS-13-dependent proteolysis of VWF in vitro and in vivo. These results suggest that FVIII and platelets may be physiological cofactors regulating VWF proteolysis. Finally, the structure–function and autoantibody mapping studies allow us to identify an ADAMTS-13 variant with increased specific activity but reduced inhibition by autoantibodies in patients with acquired TTP. Together, these findings provide novel insight into the mechanism of VWF proteolysis and tools for the therapy of acquired TTP and perhaps other arterial thrombotic disorders.


ADAMTS-13 and potential human diseases

  1. Top of page
  2. Summary
  3. ADAMTS-13 and potential human diseases
  4. Biosynthesis and secretion of ADAMTS-13
  5. Structure–function of ADAMTS-13
  6. Regulation of ADAMTS-13 function
  7. Mechanism of autoantibody inhibition
  8. Improving on nature, re-engineering ADAMTS-13
  9. Acknowledgements
  10. Disclosure of Conflict of Interest
  11. References

ADAMTS-13, first identified and cloned in 2001, is a member of the ADAMTS (A Disintegrin and Metalloprotease with ThromboSpondin type 1 repeats) family [1, 2]. It cleaves a large polymeric adhesion protein von Willebrand factor (VWF). von Willebrand factor is synthesized in vascular endothelial cells and megakaryocytes [3, 4]. The newly synthesized VWF is stored in intracellular organelles: Weibel–Palade bodies in endothelial cells and α-granules in megakaryocytes and platelets [3, 4]. VWF is released upon physiological or pathological stimulation and forms an ultra-long or ultra-large (UL) ‘string-like’ structure on the endothelial surface [5-7]. These ‘string-like’ structures are hyperactive in recruiting circulating platelets to the site of endothelial activation and/or injury. Plasma ADAMTS-13, which is primarily synthesized and released from hepatic stellate cells [8-10] and endothelial cells [11, 12], binds and cleaves cell-bound UL-VWF strings at the Tyr1605–Met1606 bond, thereby eliminating the UL-VWF from the endothelial surface and resulting in the fragmentation of the VWF strings [5-7]. In addition, ADAMTS-13 cleaves UL-VWF or large VWF in solution after being exposed to high fluid shear stress as seen in microcirculation or at the site of narrow vessels and thrombus formation after injury [13-15] (Fig. 1). Arterial shear stress induces conformational changes in soluble multimeric VWF [16-19] so that it becomes accessible by ADAMTS-13 for cleavage. The conformational changes can also be induced in vitro by an addition of a denaturant such as urea [20, 21] or guanidine-HCl [22, 23], which is the basis of various biochemical assays for plasma ADAMTS-13 activity.

image

Figure 1. ADAMTS-13 cleaves UL-VWF under various conditions. (A) ADAMTS-13 rapidly cleaves newly released UL-VWF or large VWF strings/bundles in the absence (A) and in the presence (B) of flow; ADAMTS-13 also cleaves platelet-decorated UL-VWF or large VWF strings/bundles anchored on the endothelial cell surface, in solution, and within growing thrombi under fluid shear stress (C).

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An inability to cleave cell-bound and soluble UL-VWF or large VWF in circulation because of severe deficiency of plasma ADAMTS-13 activity (< 5% of normal) results in the persistence of hyperactive VWF on endothelial cells [5, 6, 24, 25] and in circulating blood [26]. This leads to excessive platelet aggregation and disseminated VWF/platelet-rich thrombus formation [27-29], the characteristic feature of thrombotic thrombocytopenic purpura (TTP). Patients with TTP manifest by marked thrombocytopenia (platelet count, usually < 20 × 109 L−1) and microangiopathic hemolytic anemia (with low hematocrit, elevated lactate dehydrogenase, and fragmentation of red blood cells) [30-33]. Some patients may exhibit neurological signs and symptoms or renal abnormalities [30-33]. Idiopathic TTP in most adult patients is caused by an acquired deficiency of plasma ADAMTS-13 activity resulting from the formation of inhibitory autoantibodies against ADAMTS-13 [23, 34, 35]. Rarely, TTP is caused by one or several germ line mutations in the ADAMTS-13 gene [1, 34, 36-38], resulting in severe deficiency of plasma ADAMTS-13 activity at birth. Several animal models have been established for testing novel therapies. Adamts13−/− mice develop a ‘TTP-like’ syndrome only after a trigger such as shiga toxin [39, 40] or recombinant VWF challenge [41]. However, in baboon, TTP syndrome occurs subsequently after complete inhibition of plasma ADAMTS-13 activity by an intravenous administration of anti-ADAMTS-13 monoclonal antibodies against the metalloprotease domain without additional triggers [42].

The importance of discovering ADAMTS-13 extends beyond its association with the potentially fatal TTP syndrome (Table 1). Studies have demonstrated that reduced plasma ADAMTS-13 activity and increased plasma VWF (the only known substrate for ADAMTS-13) are risk factors for the development of myocardial infarction [43-45], ischemic stroke [46-48], pre-eclampsia [49], and malignant (or cerebral) malaria [50-54]. Moreover, Adamts13−/− mice demonstrate an increased (approximately 2- to 5-fold) area of atherosclerotic lesion en face and increased macrophage infiltration as compared to those in WT mice in an ApoE−/− background [55, 56]. More recently, studies have demonstrated that Adamts13−/− mice exhibit an increase in infarct sizes in the myocardium [57-59] and brain [47, 60] after ischemic/perfusion injury. An infusion of recombinant human ADAMTS-13 into Adamts13−/− mice significantly attenuates infarct sizes [47, 60]. These findings indicate that ADAMTS-13 offers systemic protection against ischemic myocardial and cerebral infarctions. Therefore, an investigation of the biosynthesis, structure–function relationship, and cofactor-dependent regulation of ADAMTS-13 protease will provide novel tools for diagnosis and treatment of many potentially fatal human diseases.

Table 1. ADAMTS-13 deficiency and potential human diseases
Diseases associatedSpeciesADAMTS-13 statusReferences
  1. TTP, thrombotic thrombocytopenic purpura; H, human; B, baboon; M, mouse; R, rat.

TTPH< 5% [1, 20, 22, 33]
MNull [39, 40]
B< 5% [42]
Ischemic strokeHReduced [43, 48]
MNull [46, 47, 60]
Myocardial infarctionHReduced [43-45]
MNull [56, 58]
AtherosclerosisHReduced [48]
MNull [55, 56]
Cerebral malariaHReduced [50-54]
PreeclampsiaHReduced [49]

Biosynthesis and secretion of ADAMTS-13

  1. Top of page
  2. Summary
  3. ADAMTS-13 and potential human diseases
  4. Biosynthesis and secretion of ADAMTS-13
  5. Structure–function of ADAMTS-13
  6. Regulation of ADAMTS-13 function
  7. Mechanism of autoantibody inhibition
  8. Improving on nature, re-engineering ADAMTS-13
  9. Acknowledgements
  10. Disclosure of Conflict of Interest
  11. References

ADAMTS-13 is primarily synthesized in the liver of humans, mice, and rats [1, 2, 8-10]. The mRNA encoding the full-length ADAMTS-13 (approximately 4.3 kb) is detected only in the liver by Northern blotting analysis [1, 2]. However, a truncated form of ADAMTS-13 mRNA (approximately 2.4 kb) is found in other tissues such as placenta and skeletal muscle by the same method [2]. Using reverse polymerase chain reaction (PCR), fragments of ADAMTS-13 mRNA are amplified in many tissues including the kidneys, pancreas, spleen, thymus, prostate, testis, ovary, small intestine, colon, and peripheral blood leukocytes [1, 61]. Whether it is synthesized in endothelial cells or parenchymal cells in these tissues remains to be determined.

In the liver, ADAMTS-13 is localized to the hepatic stellate cells (HSCs) residing in the interstitial area of human [9], mouse [10], and rat [8] livers. ADAMTS-13 mRNA and protein expression in the rat HSCs are dramatically up-regulated upon activation by mechanics and inflammatory cytokines such as transforming growth factor-β (TGF-β) in vitro and in vivo [8], suggesting that ADAMTS-13 may play a role in tissue remodeling after injury. In addition, ADAMTS-13 protein produced in the HSCs may diffuse into capillaries and enters into the blood stream, thereby regulating plasma levels of ADAMTS-13 activity. The evidence supporting this hypothesis include (i) reduced plasma ADAMTS-13 activity after a partial hepatectomy in humans [62] or after treatment with dimethylnitrosamine, which damages stellate cells in rats [63], and (ii) increased expression of ADAMTS-13 in HSCs and elevated plasma ADAMTS-13 activity upon activation in rat models of cholestasis and steatohepatitis [64].

ADAMTS-13 mRNA and protein have also been detected in vascular endothelial cells in vitro and in vivo [11, 12, 65]. It is estimated that unstimulated human umbilical vessel endothelial cells (HUVECs) in culture produce approximately 1 ng ADAMTS-13 per milliliter of conditioned medium every 60 min. The amount of ADAMTS-13 is approximately 100-fold less than VWF (100 ng mL−1) produced by HUVECs under the same conditions [65]. Immunohistochemistry demonstrates that ADAMTS-13 is not co-localized with VWF in Weibel–Palade bodies [11, 12, 65], suggesting that ADAMTS-13 is constitutively secreted from cells.

The function of ADAMTS-13 synthesized in the endothelium is not fully understood. While endothelial cells produce trace amounts of ADAMTS-13 in culture, their massive surface coverage suggests a potentially substantial contribution of the endothelium-derived ADAMTS-13 to plasma ADAMTS-13 activity. In addition, ADAMTS-13 released from endothelial cells may cleave newly formed UL-VWF strings on the cell surface, providing an additional mechanism to maintain a VWF-free surface [65-68]. Moreover, Lee et al.[69] have demonstrated that ADAMTS-13 has either pro-angiogenic or anti-angiogenic effects depending on the cellular environments. On the one hand, treatment of HUVECs with recombinant ADAMTS-13 results in dramatically increased tube formation and cell migration, suggesting enhanced angiogenesis. On the other hand, when vascular endothelial growth factor (VEGF) is present in the culture medium, ADAMTS-13 inhibits VEGF-induced angiogenic activity. This anti-angiogenic effect is reversed by pre-incubation of ADAMTS-13 with a polyclonal antibody against the C-terminal TSP1 5–7 repeats of ADAMTS-13 [69], suggesting a role of TSP1 repeats in mediating the pro- and anti-angiogenic effects.

A small quantity of ADAMTS-13 mRNA and protein is detectable in human megakaryocytes and platelets [70, 71]. One study shows that platelets contain < 160 ng ADAMTS-13 per 1 × 108 platelets [70]. The amount of ADAMTS-13 produced in platelets may be overestimated. The biological function of platelet-derived ADAMTS-13 remains unknown. Preliminary study from our laboratory, presented in the 54th Annual Meeting of the American Society of Hematology, demonstrated that transgenically overexpressed ADAMTS-13 in the platelets of Adamts13−/− mice is releasable upon activation by thrombin and collagen, as well as during the thrombus formation after injury with 10% ferric chloride [72]. The secreted human ADAMTS-13 was able to dramatically inhibit thrombus growth in mesenteric arterioles after oxidative injury and protects Adamts13−/− mice from VWF- and shiga toxin-induced ‘TTP-like’ syndrome [72]. These results suggest that platelet-derived ADAMTS-13 may be biologically important. The release of ADAMTS-13 at the site of thrombus formation may offer a novel treatment for acquired TTP with inhibitors. A similar strategy has been reported for the treatment of hemophiliacs with inhibitors with success in murine models [73-75].

Structure–function of ADAMTS-13

  1. Top of page
  2. Summary
  3. ADAMTS-13 and potential human diseases
  4. Biosynthesis and secretion of ADAMTS-13
  5. Structure–function of ADAMTS-13
  6. Regulation of ADAMTS-13 function
  7. Mechanism of autoantibody inhibition
  8. Improving on nature, re-engineering ADAMTS-13
  9. Acknowledgements
  10. Disclosure of Conflict of Interest
  11. References

ADAMTS-13 shares similar domain structure as compared to other ADAMTS family proteases, comprising a signal peptide, a propeptide, a metalloprotease, a disintegrin-like domain, first thrombospondin type 1 repeat (TSP1), and Cys-rich and spacer domains. The more distal C-terminus contains seven additional TSP1 repeats and two CUB domains (Fig. 2). The function of each domain of ADAMTS-13 in its biosynthesis, secretion, and proteolytic activity has been extensively studied in recent years.

image

Figure 2. Domain organization and partial crystal structure of ADAMTS-13. On the left, the domain organization of human mature ADAMTS-13 is shown, which consists of a metalloprotease domain (M), a disintegrin-like domain (D), the first TSP1 repeat, a Cys-rich domain (C), and a spacer domain (S). In addition, the C-terminus contains seven more TSP1 repeats [2-8] and two CUB domains (C1 and C2). On the right, the surface and carton presentation of the crystal structure of ADAMTS-13 disintegrin-like domain (Dis), first TSP1 repeat (TSP1), and Cys-rich (Cys) and spacer domain (Spa) in addition to a modeled metalloprotease domain (based on the metalloprotease domains of ADAMTS-4 and ADAMTS-5).

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Unlike the propeptides of other ADAMTS or ADAM family proteases (typically approximately 200 amino acid residues) [76, 77], the ADAMTS-13 propeptide is exceptionally short (only approximately 41 residues). While the propeptides of other ADAMTS proteases function as molecular chaperones to assist protein folding and maintain the latency of the protease by a mechanism of ‘cysteine-switch’, the ADAMTS-13 propeptide is not required for its secretion and activation. Recombinant human ADAMTS-13 expressed in cells with or without a propeptide secrets normally and is able to cleave VWF substrates with similar efficacy [78].

The metalloprotease domain of ADAMTS-13 has the expected hallmarks of the reprolysin or adamalysin family of metalloproteases, which include three histidine residues that coordinate the essential Zn2+ ion in the sequence HEXXHXXGXXHD (Fig. 3) [2]. In addition, three putative Ca2+-binding sites have been postulated based on modeling the metalloprotease domain of ADAMTS-4 and ADAMTS-5 [79] (Fig. 3). The first putative Ca2+-binding site comprises amino acid residues Glu83, Asp173, Cys281, and Asp284 that are broadly conserved among ADAMTS and other metalloproteases and appear to mediate low-affinity Ca2+ binding. The second putative Ca2+-binding site consists of residues Glu164 and Asp166 in conjunction with one or more of residues Asn162, Asp165, and Asp168. Mutations at the second putative site have no effect on the Ca2+-dependent ADAMTS-13 activity. The third site is predicted to include residues Asp187 and Glu212 in conjunction with Asp182 or Glu184 (Fig. 3). Mutations at this site dramatically reduce Ca2+-induced ADAMTS-13 activity, suggesting that the residues at the third site play an important role in high-affinity Ca2+ binding and proteolytic activity [79].

image

Figure 3. Zinc- and calcium-binding sites in the metalloprotease domain of ADAMTS-13. (A) The ADAMTS-13 metalloprotease domain with an active site and three putative calcium-binding sites; (B) Three histidine residues and one glutamic acid coordinate the zinc binding.

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Like many clotting factors, exosite interaction appears to govern enzymatic activity and substrate specificity. The ADAMTS-13 metalloprotease domain alone has no or little proteolytic activity toward VWF substrates. The proteolytic activity increases as more and more non-catalytic domains are sequentially added to the metalloprotease domain [80-83]. As shown in the model of MDTCS fragments based on the crystal structure of ADAMTS-13-DTCS [84] and the metalloprotease domains of ADAMTS-1 [85], ADAMTS-4, and ADAMTS-5 [86] (Fig. 2), the metalloprotease and disintegrin-like domain (MD) appear to be an inseparable functional unit [80, 81, 87, 88]. Experimental data also show that the addition of the disintegrin-like domain to the metalloprotease domain significantly increases cleavage efficiency and specificity [80, 87]. ADAMTS-13 variants lacking the disintegrin-like domain [80, 81, 87] or carrying point mutations in the variable regions of disintegrin-like domain (Arg349Ala and Leu350Gly) [87] have dramatically reduced proteolytic activity toward both peptide substrates and multimeric VWF, suggesting the importance of the disintegrin-like domain in substrate recognition. Further studies demonstrate that residues Arg349 and Leu350 of the ADAMTS-13 disintegrin domain may interact with residues Asp1614 and Ala1612 in the central A2 domain of VWF. Such interactions appear to assist in positioning the Tyr1605–Met1606 bond into the active-site cleft, thereby markedly affecting the rate constant (Km) and catalytic efficiency (kcat) for substrate proteolysis [87, 89]. More recently, Xiang et al. [89] demonstrated that VWF Leu1603 (P3), Tyr1605, and Asp1614 appear to make direct contact with Leu198, Val195, and Arg349 in ADAMTS-13, respectively. Therefore, the disintegrin domain, working in concert with other non-catalytic domains, ensures that the scissile bond is brought into position over the active center for cleavage to occur.

Much of our and others' attention has been focused on the role of the Cys-rich and spacer domains of ADAMTS-13 in substrate recognition [80-83, 88, 90]. ADAMTS-13 variants lacking both the Cys-rich and spacer domains or the spacer domain alone exhibit only minimal activity toward peptidyl substrates but have nearly no activity toward cell-bound UL-VWF [66, 91] and soluble VWF under various conditions [18, 80, 82, 83]. Others and we have shown that proteolytic cleavage of a peptidyl substrate increases as function of each of the non-catalytic domains (DTCS) is added to the metalloprotease domain [80, 81, 83]. However, further addition of the TSP1 2-8 and CUB domains does not increase proteolytic activity. These results suggest that the exosite interaction between the ADAMTS-13-DTCS domains and VWF-A2 domain is critical for proteolysis of VWF. These non-catalytic (DTCS) domains of ADAMTS-13 are shown to directly interact in a linear fashion with various segments in the central VWF-A2 between residues Asp1614 and Arg1668 [81, 87, 92].

A replacement of the TCS domains of ADAMTS-5, a closely related member of ADAMTS proteases, with those of ADAMTS-13 alters ADAMTS-5 substrate specificity [92]. ADAMTS-5 is not known to cleave VWF, but a chimeric variant that consists of the metalloprotease and disintegrin domains like of ADAMTS-5 (MD5) and three non-catalytic TCS domains of ADAMTS-13 (TCS13) (MD5/TCS13) is able to cleave the Glu1615–Ile1616 bond of VWF domain A2 in peptide substrates or VWF multimers that has been sheared [92]. However, this cleavage site is no longer at the Tyr1605–Met1606 bond [92], further confirming the critical role of each of non-catalytic domains of ADAMTS-13 in substrate specificity.

Further sequence analysis of the spacer domains from ADAMTS-13 and other ADAMTS family proteases has allowed us to identify several potential exosites that are highly conserved in human, murine, and zebrafish ADAMTS-13 but absent in the other members of the ADAMTS family (Fig. 4B) [93]. One of these exosites, also described as exosite 3, comprises amino acid residues Tyr658, Arg659, Arg660, Tyr661, and Tyr665 (in conjunction with two surrounding residues Arg568 and Phe592) (Fig. 4C and 4D) [84, 93]. A deletion of residues Arg659-Glu664 (Δ6aa) or a substitution of residues Arg659, Arg660, and Tyr661 with alanine dramatically reduces proteolytic cleavage of various VWF substrates [93, 94].

image

Figure 4. Sequence alignment of the spacer domains of ADAMTS-13 and other ADAMTS proteases. (A) Domain organization of ADAMTS-13; (B) Sequence alignment of a partial spacer domain (Arg629-Lys681) from human (h), murine (m), and zebrafish (z) ADAMTS-13 (A13), as well as a partial spacer domain of various ADAMTS family proteases (AD1-AD20). Boxed area in pink presents a region (exosite 3) that is highly conserved in the ADAMTS-13 spacer domain but absent in the spacer domain in other ADAMTS proteases. C and D are the surface and ribbon representation of exosite 3, respectively.

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Additional evidence to support the physiological role of exosite 3 comes from our mouse models of thrombosis. The ADAMTS-13-Δ6aa variant exhibits dramatically reduced antithrombotic activity compared with WT-ADAMTS-13 in the mesenteric arteriolar occlusion assay in Adamts13−/− mice [18]. Together, these data suggest the critical role of exosite 3 in the spacer domain in substrate recognition and proteolysis of VWF in vitro and in vivo.

ADAMTS proteases have a variable number of TSP1 repeats that may play a role in cellular localization and substrate recognition [76, 95, 96]. All TSP1 repeats contain the sequence WXXW, which is often modified by attachment of an α-mannosyl group to the C2 atom of the first Trp [97]. Seven of the eight TSP1 repeats also contain the conserved sequence CSX(S/T)CG, in which the hydroxyamino acid at position four usually is modified by the disaccharide Glc-Fuc-O-Ser/Thr [97]. It is not clear whether these post-translational modifications play a role in ADAMTS-13 function in vivo.

The TSP1 repeats in other ADAMTS proteases participate in substrate recognition and cell surface binding. The first TSP1 repeat of ADAMTS-13 binds directly to VWF73 with a Kd of approximately 136 nmol L−1 [80]. The TSP1 5–8 repeats of ADAMTS-13 appear to bind native VWF through the D4 domain [98]. Moreover, the C-terminal TSP1 repeats of ADAMTS-13 are shown to interact with the endothelial cell surface receptor CD36 [99, 100], and such an interaction may enhance proteolytic cleavage of UL-VWF under flow conditions [101]. However, human and murine ADAMTS-13 variants lacking the C-terminal 2–8 TSP1 repeats and CUB domains (MDTCS) cleave cell-bound UL-VWF [66, 91, 102] and soluble VWF [18] with similar efficacy as does the full-length ADAMTS-13. These different conclusions may be attributed to assay sensitivity and the source of recombinant ADAMTS-13 variants. Further studies are necessary to elucidate the biological function of the TSP1 repeats.

More recently, the TSP1 repeats of ADAMTS-13 are shown to contain free thiols that may react with the free thiols on the surface of UL-VWF or plasma VWF exposed under shear stress [103, 104]. Such an interaction may prevent disulfide bond exchange and formation between two apposed VWF multimers under flow, thereby attenuating VWF-mediated platelet adhesion and aggregation [103, 104].

The CUB domains are unique to ADAMTS-13 and are not found in other ADAMTS and ADAM proteases [76, 77, 96]. The role of the ADAMTS-13 CUB domains is not fully understood. Recombinant CUB-1 and CUB-1+2 domains or synthetic peptides derived from the CUB-1 domain of ADAMTS-13 partially block proteolytic cleavage of endothelial cell-bound UL-VWF by full-length ADAMTS-13 under flow [105], suggesting that the CUB domains of ADAMTS-13 may interact with UL-VWF on the endothelial cell surface. Consistent with this hypothesis, a murine ADAMTS-13 variant lacking the CUB domains (delCUB) appears to be defective in cleaving platelet-decorated UL-VWF strings in the mesenteric arterioles of Adamts13−/− mice after ferric chloride injury [91]. However, this hypothesis possesses some challenges. A human ADAMTS-13 variant lacking the CUB domains normally cleaves the newly released UL-VWF strings in the absence of flow [66] or platelet-decorated UL-VWF strings on cultured HUVECs under flow [102]. Moreover, a similar human ADAMTS-13 variant is significantly able to attenuate the rate of thrombus growth in the mesenteric arteriolar occlusion assay [18], suggesting that the CUB domains are dispensable under these conditions. These conflicting results point to the complexity of assessing ADAMTS-13 function under more physiological conditions.

Regulation of ADAMTS-13 function

  1. Top of page
  2. Summary
  3. ADAMTS-13 and potential human diseases
  4. Biosynthesis and secretion of ADAMTS-13
  5. Structure–function of ADAMTS-13
  6. Regulation of ADAMTS-13 function
  7. Mechanism of autoantibody inhibition
  8. Improving on nature, re-engineering ADAMTS-13
  9. Acknowledgements
  10. Disclosure of Conflict of Interest
  11. References

Cofactor-dependent regulation of coagulation enzymes has been well recognized and increases the rate of an enzymatic reaction by several orders of magnitude [106, 107]. Unlike other clotting factors that are synthesized as inactive zymogens, ADAMTS-13 is secreted as a constitutively active protease [10, 11, 65, 83]. There has been no inhibitor identified to date. Plasma α2-macroglobulin inhibits many other matrix metalloproteases including ADAMTS-4, -5, -7, and -12 [108-111] but does not seem to bind and affect ADAMTS-13 activity toward VWF [83]. Therefore, ADAMTS-13 function must be regulated at the substrate level.

Newly released UL-VWF anchored on the endothelial cell membrane can be rapidly cleaved by ADAMTS-13 in the presence [5-7] or absence of flow [66, 67], suggesting that cell-bound UL-VWF is in its ‘open’ conformation. The mechanism underlying the ‘open’ conformation is not known. Once released into solution, the UL-VWF rapidly adopts a ‘closed’ conformation that becomes highly resistant to proteolysis by ADAMTS-13 in the absence of shear stress or denaturants. The soluble UL-VWF regains its sensitivity to ADAMTS-13 when exposed to high fluid shear stress (approximately 20–100 dynes cm−2) that presumably unfolds the central A2 domain of VWF [16, 17, 112]. Such high shear conditions can be found in vivo in narrowed or branching vessels, arteries, arterioles, and microcirculation. The increased VWF proteolysis by ADAMTS-13 and the reduction in the plasma VWF activity-to-antigen ratio [113] correlate with the severity of aortic stenosis [15, 114]. Surgical correction normalizes plasma VWF multimer distribution [15, 115].

Arterial shear stress can be simulated in vitro using a cone plate viscometer [116, 117], a bench-top mini-vortex [98, 112, 118], and a microfluidic system [119-121] that generates laminar flow. Under mechanically induced shear stress, proteolytic cleavage of multimeric VWF by recombinant ADAMTS-13 increases as a function of increasing shear rate (or shear stress), incubation time, and concentrations of ADAMTS-13 enzyme [112]. The mechanical force-induced cleavage of an A1A2A3 tri-domain molecule [19] or the A2 domain of VWF [122, 123] has also been demonstrated. Together, these findings suggest that fluid shear stress plays a critical role in regulating proteolytic cleavage of soluble VWF by ADAMTS-13.

In addition to shear stress, coagulation factor VIII (FVIII), which binds VWF with high affinity (KD, 0.25–0.5 nmol L−1), may alter the domain–domain interaction of the neighboring A1A2A3 of VWF and regulate proteolytic cleavage of the A2 domain by ADAMTS-13. To test this hypothesis, recombinant FVIII at various concentrations (0–20 nmol L−1) was incubated for 30 min with plasma-derived or recombinant multimeric VWF prior to incubation with recombinant ADAMTS-13. After 2-min incubation under constant vortexing (2500 rpm, approximately 28.5 dynes cm−2), the proteolytic cleavage product (approximately 350 kDa) increases as a function of increasing concentrations of FVIII [118]. In the presence of 20 nmol L−1 FVIII, the maximal increase in the cleavage product formation approaches approximately 10- to 12-fold of that with VWF alone. The rate-enhancing effect of FVIII on VWF proteolysis was detected under fluid shear stress but not under static/denaturing conditions [118], suggesting that the binding of FVIII to VWF may facilitate the unfolding processes of the VWF-A2 domain under the shear conditions.

Structure–function analysis demonstrates that the B-domain-deleted FVIII variant (FVIII-SQ) exhibits a similar rate-enhancing effect on proteolysis of VWF by ADAMTS-13 as does full-length FVIII [118, 124]. However, a FVIII variant lacking the acidic (a3) region that contains a major VWF-binding site (FVIII-2RKR) has no effect under the same conditions [118, 124]. Interestingly, a light chain of FVIII (FVIII-LC), despite a 10-fold reduction in its binding affinity to VWF, is sufficient for accelerating the cleavage of VWF to a similar extent as are wild-type FVIII and FVIII-SQ[124], suggesting that binding of FVIII to VWF through its light chain mediates this cofactor activity.

These biochemical findings are further corroborated with those obtained in vivo in a murine model. Hydrodynamic injection is a commonly used method to instantly transfect hepatocytes with plasmids of interest. This maneuver also activates endothelial cells, triggering the release of UL-VWF into plasma in mice. When injected with PBS alone, plasma ratios of high- to low-molecular-weight VWF multimers in fVIII−/− mice are higher than those in the fVIII−/− mice reconstituted with a plasmid encoding FVIII-SQ or FVIII-LC [124], suggesting that the expression of a functional VWF-binding FVIII variant eliminates the accumulation of UL-VWF multimers under (patho)physiological conditions.

Additional evidence to support the physiological role of FVIII-dependent regulation of VWF proteolysis by ADAMTS-13 comes from our recent findings (presented in part at the ASH meeting, 2009) [125]. The result demonstrated that type 2N VWF variants that exhibit a moderate to severe defect for FVIII binding were also compromised in accelerating cleavage of VWF by ADAMTS-13 in the presence of FVIII under shear stress [125]. Together, our findings support the critical role of FVIII as a physiological cofactor regulating proteolytic cleavage of VWF by ADAMTS-13. Such cofactor activity is dependent on the interaction between the light chain of FVIII and the D'D3 domains of VWF.

Platelet glycoprotein 1bα (GP1bα) also binds VWF with high affinity. Studies have demonstrated that an addition of formalin-fixed, lyophilized or fresh platelets and soluble GP1bα to multimeric VWF increases proteolytic cleavage by ADAMTS-13 under static [116, 126] or fluid shear conditions [117]. Ristocetin, an antibiotic that binds the A1 domain of VWF close to the site that GP1bα binds, also enhances cleavage of multimeric VWF by ADAMTS-13 [117, 127]. These results suggest that the interaction between platelet GP1bα (or ristocetin) and the A1 domain affects the accessibility of the A2 domain by ADAMTS-13. Interestingly, ristocetin alleviates the requirement of FVIII to enhance the cleavage of VWF by ADAMTS-13 [117], while binding of platelet GP1bα to VWF enhances the effect of FVIII or vice versa as previously demonstrated. In the presence of physiological concentrations of platelets (150 × 103 μL−1), the C50 shifts to the left (from 5 to 0.5 nmol L−1) [117]. These results suggest that FVIII and platelet GP1b have synergistic effects to enhance VWF proteolysis by ADAMTS-13 under fluid shear stress. It has been postulated that the binding of FVIII to the D'D3 domain of VWF may result in large-scale conformational changes in the VWF multimers, such as pulling away the D'D3 domain from its neighboring A1 or A2 domain under shear stress. Similarly, binding of platelets or soluble GP1bα to the VWF-A1 domain may expose the A2 domain for cleavage. Two or more platelets bound on each side of the scissile bond may dramatically increase the peak tensile force exerted on the central A2 domain [128], which enhances A2 domain unfolding and proteolysis by ADAMTS-13, demonstrated by single molecule experiments [19, 122, 123].

Mechanism of autoantibody inhibition

  1. Top of page
  2. Summary
  3. ADAMTS-13 and potential human diseases
  4. Biosynthesis and secretion of ADAMTS-13
  5. Structure–function of ADAMTS-13
  6. Regulation of ADAMTS-13 function
  7. Mechanism of autoantibody inhibition
  8. Improving on nature, re-engineering ADAMTS-13
  9. Acknowledgements
  10. Disclosure of Conflict of Interest
  11. References

Idiopathic TTP in adults is mainly caused by a severe deficiency of plasma ADAMTS-13 activity due to immunoglobulin G (IgG)-type antibodies. Depending on the assays, the inhibitory antibodies are detected in 44–100% of acquired idiopathic TTP patients with a severe deficiency of plasma ADAMTS-13 activity [129]. With more sensitive assays such as an enzyme-linked immunosorbent assay (ELISA) [130, 129] or flow cytometry–based technology [131], anti-ADAMTS-13 IgGs can be detected in all patients with TTP who have a severe deficiency of plasma ADAMTS-13 activity [129]. Antibody mapping and profiling reveal that anti-ADAMTS-13 IgG1 and IgG4 predominate in the plasma of patients with acquired TTP [132, 133], and nearly all anti-ADAMTS-13 IgGs bind the Cys-rich and spacer domains, particularly the spacer domain [35, 94, 134-138]. Other ADAMTS-13 domains including the propeptide, metalloprotease domain, disintegrin domain, first TSP1 repeat, more distal TSP1 repeats, and CUB domains are less reactive with the autoantibodies [35, 134, 138]. Further analysis demonstrates that the major antigenic epitopes are localized to residues Tyr572-Asn579 [137], Val657-Gly666 [94, 137], and Gly662-Val687 [139]. A majority of patients with TTP (90%) lose reactivity toward ADAMTS-13 following the substitution of residues Arg568, Phe592, Arg660, Tyr661, and Tyr665 in exosite 3 of the spacer domain [138]. These residues have been demonstrated to play a critical role in substrate recognition and proteolysis of VWF under various conditions [93, 94]. Therefore, it is conceivable that the binding of anti-ADAMTS-13 autoantibodies to this region blocks substrate binding and its proteolytic function.

The mechanism of how patients develop autoantibodies against ADAMTS-13 protease is not known. Female predominance [33, 35] and autoantibodies observed in identical twin sisters [140] suggest a genetic predisposition. A recent study demonstrates an overrepresentation of the HLA-DRB1*11 allele in patients with acquired TTP [141], consistent with the hypothesis. ADAMTS-13 is efficiently internalized by immature dendritic cells (antigen-presenting cells) through the surface macrophage mannose receptor [142, 143]. Interestingly, dendritic cells from HLA-DRB1*11 donors pulsed with higher concentrations of ADAMTS-13 in culture present derivatives of a single CUB-2-derived peptide, suggesting that functional presentation of CUB-2-derived peptides on HLA-DRB1*11 may contribute to the onset of acquired TTP by stimulating low-affinity self-reactive CD4+ T cells [142, 144]. Moreover, bacterial or viral infections often precede the acute epitope of initial or recurrent TTP [145-147]. This indicates that infections may act as a trigger boasting the production of autoantibody production or substrate release from activated endothelium. However, one cannot exclude the possibility of a ‘by-stander’ or molecular mimic hypothesis in which antibodies against microbes may cross react with ADAMTS-13.

Improving on nature, re-engineering ADAMTS-13

  1. Top of page
  2. Summary
  3. ADAMTS-13 and potential human diseases
  4. Biosynthesis and secretion of ADAMTS-13
  5. Structure–function of ADAMTS-13
  6. Regulation of ADAMTS-13 function
  7. Mechanism of autoantibody inhibition
  8. Improving on nature, re-engineering ADAMTS-13
  9. Acknowledgements
  10. Disclosure of Conflict of Interest
  11. References

Others and we have demonstrated that the spacer domain [35, 94, 134, 136-138], particularly exosite 3 (Figs 2 and 4), is the major target of anti-ADAMTS-13 autoantibodies in patients with acquired TTP [94, 138]. Autoantibody binding to this region is expected to block substrate binding and inhibit proteolytic activity of ADAMTS-13. Replacement of the residues in exosite 3 with alanine nearly completely abolishes anti-ADAMTS-13 IgG binding, but also significantly impairs proteolytic activity of ADAMTS-13. The loss-function-ADAMTS-13 mutants have no value for therapy. We sought to subtly modify these residues in exosite 3 by changing Arg to Lys or vice versa in hopes of eliminating autoantibody binding but retaining ADAMTS-13 activity. Through site-directed mutagenesis, we have created and tested a panel of ADAMTS-13 variants for antibody binding and proteolysis. The results show that a substitution of 4–5 residues in exosite 3 generates ADAMTS-13 variants (M4: R660K/F592Y/R568K/Y661F and M5: R660K/F592Y/R568K/Y661F/Y665F) with increased specific activity toward FRETS-VWF73 and multimeric VWF by 4- to 5- and 10- to 12-fold, respectively (Fig. 4) [148]. More interestingly, these variants were more resistant than WT and M1–M3 (with one to three residues mutated) to inhibition by anti-ADAMTS-13 autoantibodies from a majority of patients with acquired TTP [148]. The reduction of the M4 and M5 to inhibition by autoantibodies is correlated with the impaired binding of anti-ADAMTS-13 IgG to the variants [148]. Together, our findings indicate that it is possible to re-engineer ADAMTS-13 protease to improve specific activity in the presence of autoantibodies, which may offer therapeutic benefits to acquired TTP with inhibitors.

In summary, tremendous progress has been made in the past decade toward our understandings of biosynthesis, structure–function relationship, and cofactor-dependent regulation of ADAMTS-13 protease. These advances provide invaluable information for our understandings of the mechanisms of TTP and other atherothrombotic disorders, as well as inflammatory diseases. Therefore, novel diagnostic tools and therapeutics may be developed for managing these potentially fatal diseases.

Acknowledgements

  1. Top of page
  2. Summary
  3. ADAMTS-13 and potential human diseases
  4. Biosynthesis and secretion of ADAMTS-13
  5. Structure–function of ADAMTS-13
  6. Regulation of ADAMTS-13 function
  7. Mechanism of autoantibody inhibition
  8. Improving on nature, re-engineering ADAMTS-13
  9. Acknowledgements
  10. Disclosure of Conflict of Interest
  11. References

The work presented in the review article was supported by grants from The Children's Hospital of Philadelphia, The University of Pennsylvania, the National Blood Foundation, the American Heart Association (04265532U and 0940100N), and the National Institute of Health (HL-079027 and HL-074124).

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  1. Top of page
  2. Summary
  3. ADAMTS-13 and potential human diseases
  4. Biosynthesis and secretion of ADAMTS-13
  5. Structure–function of ADAMTS-13
  6. Regulation of ADAMTS-13 function
  7. Mechanism of autoantibody inhibition
  8. Improving on nature, re-engineering ADAMTS-13
  9. Acknowledgements
  10. Disclosure of Conflict of Interest
  11. References
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