Left ventricular assist devices (LVADs) provide cardiac support for patients with end-stage heart disease, but device-related hemostatic complications remain common and are associated with poor outcomes. Bleeding is most common and is primarily found in the gastrointestinal track at the site of an arteriovenous malformation,1,2 leading to the hypothesis that it is caused by the loss of large von Willebrand factor (VWF) multimers3–5 in a condition called “acquired von Willebrand syndrome” (AVWS).3,4 Acquired von Willebrand syndrome is widely considered to be caused by excessive VWF cleavage by the metalloprotease ADAMTS-13 (a disintegrin and metalloprotease with thrombospondin type-1 repeats 13).5,6 Von Willebrand factor cleavage can indeed be induced by elevated shear stress7–9 similar to that of an LVAD-driven blood flow.10 However, several lines of evidence suggest that excessive cleavage by ADAMTS-13 is not the sole cause of AVWS. First, there is no direct evidence that VWF multimers are excessively cleaved by ADAMTS-13 in these patients. Second, the loss of large VWF multimers is observed in nearly all patients, but only 11–30% of them bleed.1,11 Third, high shear stress induce not only VWF cleavage but also VWF binding to platelets (PLTs) and VWF-mediated PLT aggregation.12–14 These shear stress–activated VWF multimers could bind to PLTs and, thus, be removed from the circulation, leading the consumptive loss of large VWF multimers. Using a newly synthesized antibody specific for cleaved VWF, we have recently demonstrated that the two pathways could cause the loss of large VWF multimers in patients on LVAD support: excessive cleavage (loss-of-function) and shear-induced VWF activation (gain-of-function).15 Although both processes can result in AVWS, they can lead to distinct hemostatic defects that require different treatments. A technical limitation of the antibody study is its inability to accurately quantify VWF cleavage. To address this limitation, we have developed a targeted mass spectrometry to quantify VWF cleavage by ADAMTS-13 in plasma samples from patients on LVAD support.
Materials and Methods
Blood samples were collected from 14 patients (age: 40–69 years; 50% females) before and 3 months after LVAD implants. The recruitment was approved by the institutional review boards of the Texas Heart Institute and the Bloodworks Research Institute. All patients had New York Heart Association class IV symptoms and received a HeartMate II LVAD (Thoratec, Pleasanton, CA). Patients with malignancy, autoimmune disease, or were at a hypercoagulable state were excluded.
Selected reaction monitoring (SRM) was used to measure VWF and its rate of cleavage in plasma samples. Von Willebrand factor levels were measured by four tryptic VWF peptides: EGGPSQIGDALGFAVR, VTVFPIGIGDR, and ILAGPAGDSNVVK in the A3 domain and SGFTYVLHEGECCGR in the C domain. These peptides were chosen for being proteotypic and for having a unique signature (m/z ratio) in the proteome for every Q1/Q3 pair from the peptides, as determined by the in-house human proteomics database SRMAtlas (www.srmatlas.org).16 In addition, the ADAMTS-13–cleaved peptide M1606VTGNPASDEIK was detected. Internal peptide standards for all five VWF peptides were synthesized with heavy isotopic lysine (13C615N2) or arginine (13C615N4) at the C-termini (Thermo Fisher Scientific, Rockford, IL). The total peak area and the ratio of plasma peptide and its synthesized heavy counterpart were normalized to quantify VWF and its cleavage.
Von Willebrand factor purified from human cryoprecipitate and a recombinant human VWF A1-3 domain peptide was cleaved by ADAMTS-13 in vitro6 and tested as a control. Von Willebrand factor cleavage was also measured by immunoblots for data validation. Von Willebrand factor antigen was measured by a commercial enzyme-linked immunosorbent assay (ELISA).15
Results and Discussion
The baseline demographic, diagnosis, and laboratory results of patients examined in this study are presented in Table 1. Clinical indexes reported here have been selected to estimate vital organ functions: hemopoietic cells by hemoglobin (also for hemolysis) and white blood cell counts, hemostasis by PLTs and prothrombin time, the nutritional status by albumin, the renal function by creatinine, and bilirubin (total and indirect) as the marker of baseline liver function and index of preimplant right-sided dysfunction. Normal control plasma was pooled from 61 healthy control subjects, allowing us to cost–effectively test a large number of donors to cover the full range of normal values. The four uncleaved VWF peptides profiled in Figure 1 (Supplemental Digital Content, https://links.lww.com/ASAIO/A170) were detected at comparable levels in all plasma samples (Figure 1A) with a reproducibility of 96.8%. The levels of the four peptides in the baseline samples before LVAD implantation were significantly higher than those in healthy control (Figure 1A, black bars). They were reduced after LVAD implantation, but remained higher than normal controls (Figure 1A, white bars), consistent with our recent report.15 The mass spectrometry results were validated by their close association with VWF antigen levels measured by ELISA (Figure 1B; r2 = 0.697; p < 0.001). The detection of these peptides was also validated using a recombinant VWF A1-3 domain polypeptide that was digested by ADAMTS-13 (Figure 2, Supplemental Digital Content, https://links.lww.com/ASAIO/A171). In contrast to the readily detection of these four VWF peptides, the cleaved peptide MVTGNPASDEIK (Figure 1C) was detected only after VWF was concentrated 20-fold by immunoprecipitation with a polyclonal VWF antibody. Consistent with SRM data, cleaved VWF was also primarily detected by immunoblots in plasma spiked with cleaved VWF (Figure 1D). Furthermore, M1606VTGNPASDEIK had been detected only in the methionine-oxidized form (molecular mass: 1277.41 vs. 1261.41 for oxidized and nonoxidized peptides, respectively [Figure 3, Supplemental Digital Content, https://links.lww.com/ASAIO/A172]) in samples from the patients and from healthy subjects. The finding suggests that the Met1606 is highly sensitive to oxidation during sample processing and that the synthesized heavy peptide should be oxidized to detect and quantify VWF cleavage. Together, these data demonstrate that SRM reliably and quantitatively detected VWF antigen and its cleavage by ADAMTS-13, simultaneously.
Using this newly established SRM, we determined that the plasma levels of the cleaved M1606VTGNPASDEIK peptide in samples collected 3 months after LVAD implantation varied significantly, but were mostly below the level of normal plasma pooled from 61 healthy subjects (Figure 2A). To adjust for high VWF antigen in these patients (Figure 1B), we defined VWF cleavage as the ratio of M1606VTGNPASDEIK to ILAGPAGDSNVVK. This rate varied significantly in the post-LVAD samples, accounting for 0.23–2.5% of total VWF as compared to 1.26% ± 0.36% in normal plasma, even though the loss of large VWF multimers was found in 91.3% of patients as we have recently reported.15 The findings suggest that 1) circulating VWF was minimally cleaved in normal subjects and 2) there was no drastic increase in VWF cleavage in post-LVAD samples. These findings raise a possibility that VWF was cleaved by ADAMTS-13 primarily to be released from its anchorage to endothelial cells as we previously shown.17 Although further experiments are required to determine whether such small changes in VWF cleavage are sufficient to alter VWF reactivity, a subgroup analysis did find that VWF cleavage in post-LVAD samples was increased in patients in whom bleeding was developed and was mostly reduced in patients in whom thrombosis was developed (Figure 2B). Because this study was primarily focused on developing a quantitative method to measure VWF cleavage, it had a limited number of patients. This small patient cohort is not powered to determine the association of VWF cleavage with the development of hemostatic complications in patients on LVAD support.
In summary, we have developed a reliable mass spectrometry–based method to quantify VWF cleavage by ADAMTS-13 in plasma. We made four novel observations. First, we provide first quantitative evidence that cleaved VWF accounts for less than 2% of total VWF in circulation. This finding suggests that the multimer patterns of plasma VWF are not entirely resulted from ADAMTS-13 cleavage, but could also attribute to the redox regulation of the intramultimer disulfide bounds as previously reported.18–20 Second, VWF cleavage was reduced in 71.4% of heart failure patients before LVAD implantation, consistent with reduced ADAMTS-13 activity in patients with myocardial infarction and inflammation.21 Third, VWF cleavage was not uniformly increased (as has been widely speculated), but rather varied significantly among patients on LVAD supports, even though AVWS is found in >90% of patients. Von Willebrand factor cleavage patterns appear to correlate with adverse events in which increased VWF cleavage mirrors bleeding, whereas decreased cleavage is more common in patients with thrombotic complications. These findings, together with our recent report,15 suggest that the LVAD-associated AVWS can be caused not only by increasing cleavage (loss-of-function) but also by shear-induced VWF binding to PLTs, and potentially to endothelial cells (gain-of-function).15 Although both processes can result in the loss of large VWF multimers, they may require different treatments. The enhancement of VWF cleavage may be more effective for patients with the gain-of-function acquired Von Willebrand syndrome (AWAS), whereas blocking VWF cleavage may be necessary for patients with the loss-of-function AWAS. This SRM method can therefore be used to define the relationship among VWF cleavage, AVWS, and hemostatic complications in patients on LVAD support.
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