Identification of extant vertebrate Myxine glutinosa VWF: evolutionary conservation of primary hemostasis

Abstract

Hemostasis in vertebrates involves both a cellular and a protein component. Previous studies in jawless vertebrates (cyclostomes) suggest that the protein response, which involves thrombin-catalyzed conversion of a soluble plasma protein, fibrinogen, into a polymeric fibrin clot, is conserved in all vertebrates. However, similar data are lacking for the cellular response, which in gnathostomes is regulated by von Willebrand factor (VWF), a glycoprotein that mediates the adhesion of platelets to the subendothelial matrix of injured blood vessels. To gain evolutionary insights into the cellular phase of coagulation, we asked whether a functional vwf gene is present in the Atlantic hagfish, Myxine glutinosa We found a single vwf transcript that encodes a simpler protein compared with higher vertebrates, the most striking difference being the absence of an A3 domain, which otherwise binds collagen under high-flow conditions. Immunohistochemical analyses of hagfish tissues and blood revealed Vwf expression in endothelial cells and thrombocytes. Electron microscopic studies of hagfish tissues demonstrated the presence of Weibel-Palade bodies in the endothelium. Hagfish Vwf formed high-molecular-weight multimers in hagfish plasma and in stably transfected CHO cells. In functional assays, botrocetin promoted VWF-dependent thrombocyte aggregation. A search for vwf sequences in the genome of sea squirts, the closest invertebrate relatives of hagfish, failed to reveal evidence of an intact vwf gene. Together, our findings suggest that VWF evolved in the ancestral vertebrate following the divergence of the urochordates some 500 million years ago and that it acquired increasing complexity though sequential insertion of functional modules.

Figure 1.

Domain structure of human and hagfish VWF. (A) Schematic representation of the overall domain structure of human and hagfish VWF based on recently revised domain assignments. Shown are annotations of the furin cleavage site (solid arrow), the heparin, FVIII, GPIbα, and collagen binding sites, the RGD recognition site for GPIIb/IIIa, the ADAMTS13 cleavage site (dashed arrow), and known human exons. Note that hagfish Vwf lacks the A3 domain and the large adjacent multidomain region, D4N-VWD4-C8-4-TIL-4-C1.C, C-terminal end; N, N-terminal end. (B) Amino acid alignments of VWF sequences from varied vertebrate species at the furin cleavage site (red arrowhead). (C) Amino acid alignments of VWF sequences from varied vertebrate species at the ADAMTS13 cleavage site (red arrowhead). ADAMTS13, a disintegrin and metalloproteinase with thrombospondin motifs 13; RGD, Arg-Gly-Asp.

Figure 2.

Hagfish vwf is expressed as a single gene product. (A) PCR primer pairs A1/S1 and A2/S2 (supplemental Table 1) were used to generate DNA products from the A2 domain or from a region of the A2 domain to the C2-C3 spacer from hagfish vwf cDNA prepared by reverse transcription from hagfish heart, liver, gill, and skin RNA. Only the expected 189-bp and 333-bp PCR products were observed on agarose gel analysis. (‐‐) Control reactions without template cDNA. (B) Northern blot hybridization of hagfish RNA from heart and gill with probes that spanned either the A1-A2 region or the 3′ untranslated region (UTR) from the hagfish vwf gene (supplemental Table 1). A single 7.5-kb transcript is observed in both heart and gill.

Figure 3.

Hagfish Vwf is expressed in endothelium and peripheral blood. (A) qPCR analysis of hemostatic factors in hagfish organs and blood. mRNA expression is represented as copy number per 10⁶ 18S copies. Data are presented as mean ± standard deviation (SD) (n = 3 fish). (B) Serial sections of the body wall and muscle of hagfish stained with hematoxylin and eosin (H&E) (left) and processed for immunofluorescence staining using polyclonal anti-human VWF antibody (right). Nuclei are stained with 4′,6-diamidino-2-phenylindole (DAPI). Positive Vwf staining is observed in the aorta (*), posterior cardinal veins (arrows), capillaries of the intestinal wall (¶), and capillaries surrounding red muscle fibers (arrowheads). All stain and magnification information for photomicrographs is provided in the supplemental Material. G, gut lumen; K, kidney; N, notochord; R, red muscle fiber; W, white muscle fiber. (C) Fluorescence microscopy images of hagfish blood processed for immunofluorescence staining of Vwf using polyclonal anti-human VWF antibody. Left image (i) shows 4 Vwf-positive cells (arrows), which are smaller than neighboring Vwf-negative erythrocytes (1 of these is outlined in white). Right image (ii) is a higher-power view showing the punctate staining pattern of a Vwf-positive cell surrounded by Vwf-negative erythrocytes and spindle cells. All stain and magnification information for photomicrographs is provided in the supplemental Material. *Representative spindle cell. See supplemental Figure 4C-D for greater detail of negatively stained cells.

Figure 4.

Punctate Vwf staining and presence of WPBs in hagfish endothelial cells. Representative electron microscopy image of hagfish endothelium from aorta (A) and skeletal muscle arteriole (B) showing the presence of cigar-shaped organelles containing electron-dense tubules, consistent with WPBs. (C) Confocal laser scanning microscopy image of en face hagfish aorta processed for immunofluorescence staining of Vwf using polyclonal anti-human VWF antibody (see supplemental Figure 3 for images using hagfish monoclonal Vwf antibody). Nuclei are stained with DAPI. Note the presence of punctate staining in the endothelial cells. All stain and magnification information for photomicrographs is provided in the supplemental Material.

Figure 5.

Hagfish Vwf processing, plasma multimers, ADAMTS13 cleavage, and platelet aggregation. (A) Western blot (WB) of heart lysates from mouse or hagfish using polyclonal anti-human VWF antibody (Dako) or monoclonal anti-hagfish VWF antibody (B10), and of hagfish plasma using either monoclonal anti-hagfish Vwf antibody (B10) or polyclonal anti-hagfish Vwf antibody. Shown are bands whose sizes are consistent with mouse pro-VWF (∼300 kDa) and mature VWF (∼250 kDa), or hagfish pro-VWF (∼240 kDa) and mature VWF (∼165 kDa). (B) Human, mouse, and hagfish plasma proteins were separated on a nonreducing 1.5% agarose gel. Western blotting was carried out with polyclonal anti-human VWF antibody (Dako) alongside molecular weight marking samples of reduced and nonreduced recombinant human VWF (rhVWF) and recombinant human ADAMTS13 (rhADAMTS13). (C) Samples of hagfish Vwf from expressing CHO-cell lysates in 1.5 M urea buffer were incubated for up to 10 hours with 70 nM of full-length recombinant human ADAMTS13 with (+) or without (−) 15 mM EDTA. Note the significant reduction of the ∼240-kDa pro-Vwf band in the presence of ADAMTS13 and the appearance of a C-terminal cleavage product (∼61 kDa) detected with monoclonal anti-hagfish B10 antibody raised against C-terminal Vwf sequence. (D) Thrombocyte-rich and thrombocyte-poor plasma was prepared from ∼12 hagfish animals and measured for thrombocyte aggregation as measured by light transmission over time. Thrombocytes resuspended in thrombocyte-poor plasma were equilibrated for 2 minutes at 37°C with stirring and then incubated with varying concentrations of botrocetin (B jararaca snake venom). Thrombocyte agglutination was monitored over time (reported as minutes following addition of botrocetin) using a dual-channel optical aggregometer. Data represent the mean and SD from 3 independent experiments. Statistical analysis was carried out using paired Student t test (*P ≤ .5; **P ≤ .005). polyAB, polyclonal antibody; TBS, Tris-buffered saline; WB, western blot.

Figure 6.

Hagfish VWF forms multimers in mammalian cell culture. (A) qPCR analysis of hagfish vwf in untransfected CHO cells (CHO WT) and CHO cells stably transfected with hagfish vwf (clones 18 and 23). mRNA expression is represented as copy number per 10⁶ 18S copies. Data are presented as mean ± SD (n = 3 fish). (B) Heterologous expression of hagfish Vwf in CHO cell culture visualized by western blotting of whole-cell lysates on reducing or nonreducing SDS-PAGE with B10 anti-hagfish mAb or Dako anti-human pAb, as indicated. (C) Representative confocal laser scanning microscopy images of control CHO cells (CHO WT) or CHO cells stably transfected with hagfish Vwf (CHO18 Hagfish Vwf) processed for staining of Vwf using polyclonal anti-human VWF antibody (Dako) or monoclonal anti-hagfish Vwf antibody (B10). All stain and magnification information for photomicrographs is provided in the supplemental Material. (D) Electron micrographs of control CHO cells (CHO WT) and CHO cells expressing hagfish tissue factor pathway inhibitor (CHO Hagfish Tfpi) or hagfish Vwf (CHO18 Hagfish Vwf). Nucleus appears at the top of each cell. Note the large irregularly shaped organelles (arrowheads) in the Vwf-expressing cells which contain electron dense material.

Figure 7.

Hagfish VWF A1 and A2 domain structures. (A) Comparative model of hagfish A1 (backbone ribbon representation on surface volume renderings) in comparison with the experimentally determined human A1 structure (PDB ID 1AUQ). The structures are highly superimposable with the exception of α4, where hagfish is truncated by 4 residues, similar to known bird, amphibian, and fish sequences, impacting the length of the helix and β4α4 loop conformation. (B) Superimposition of the hagfish A1 onto experimentally determined human A1 bound to GPIbα (green ribbon) (PDB ID 3SQ0). The many amino acids shown to contribute to the GPIbα binding interface that are conserved in hagfish A1 are highlighted (gray sticks) along with those few not conserved in hagfish A1 (yellow sticks). In addition, charge is conserved between hagfish and human among 5 of the 7 electrostatically charged residues, as is the hydrophobic character of 3 of 4 residues. (C) Comparative model structure of hagfish A2 in comparison with the experimentally determined human A2 structure (PDB ID 3ZQK). Like human, hagfish A2 is distinguished from other VWA domains by a relatively long unstructured loop at helix 4 (α4). (D) Modeling of the α4-less loop environment in hagfish A2. The α3- and α5-helix dipole moments are symbolized. The strictly conserved buried hydrophobic I1622 side chain atoms, and D1620 N-cap and R1624 C-cap side chain atoms are shown (yellow sticks). Although the hagfish α4-less region contains 5 additional residues compared with human, the α4-less loop environment is well conserved.