von Willebrand disease

Von Willebrand disease (vWD) is the most common inherited disorder of hemostasis in both humans and dogs. It is due to a deficiency or abnormality in von Willebrand factor (vWf).

Production of vWf

Von Willebrand factor is a large multimeric glycoprotein that circulates non-covalently with factor VIII coagulant protein. It is produced in different cells and has different roles in hemostasis. It used to be called factor VIII-related antigen (because it is such a large protein, antibodies raised against FVIII initially only detected vWf, hence the “related antigen” moniker), however this term is completely obsolete and should be avoided.

Von Willebrand factor is produced by endothelial cells and megakaryocytes (factor VIII is produced by hepatocytes) and is stored in the alpha granules in platelets and in special organelles called Weibel-Palade bodies in endothelial cells. Both alpha-granules and Weibel-palade bodies serve as intracellular storage organelles of vWf. Dogs have a very small percentage of vWf in platelets (3%, Parker et al 1991) compared to cats (Waters et al 1989) or human beings (20%).

Von Willebrand factor is produced as a single protein chain (called a monomer), which then dimerizes within the cytoplasm of the megakaryocyte or endothelial cell. Therefore, the smallest component of vWf is a dimer. The dimer spontaneously forms long chains or polymers called multimers, which are held together by disulfide bonds. These multimers impart a very high molecular weight on vWf. The multimeric structure of vWf is important as the higher molecular weight multimers are more effective in hemostasis, so a relative deficiency of these multimers (which occurs in type II vWD) produces more severe bleeding. Von Willebrand factor is secreted as low molecular weight multimers constitutively into the extracellular matrix on the basilar surface of endothelial cells. Within the matrix, vWf is poised to bind to its ligand GP1b-IX-V on the surface of platelets, when the endothelial cells are disrupted upon vascular injury. The endothelial cells also secrete small amounts of vWf, in the form of high molecular weight multimers, constantly into plasma. Stores of vWf within Weibel-Palade bodies also consist of high molecular weight variants, which are released with stimuli, such as endothelial injury, thrombin and histamine (Lopes da Silva and Cutler 2016). As for any reaction in hemostasis, there is a reaction, or control mechanism. For vWf, the control mechanism is an enzyme called ADAMTS13 (a disintegrin-like and metalloproteinase domain with thrombospondin type-1 motif, #13), which cleaves vWf, breaking it down into smaller molecular weight multimers (De Ceunynck et al 2013).

Function of vWf

Von Willebrand factor has an essential role in primary hemostasis, being important for initiating platelet adhesion to the subendothelium in vessels with high shear rates. It also has a minor role in participating in platelet aggregation. Essentially, vWf acts like as an initial sticky tether, capturing circulating platelets and slowing their velocity in fast moving blood in vessels (such as arterioles), allowing for firmer adhesion to be mediated by slower acting integrins. The multimeric nature of vWf is important in this capturing effect (think of the unfurled protein as a string upon which platelets can transiently bind and roll), whereas compact vWf cannot facilitate initial adhesion to the same extent, presenting far fewer binding sites to platelet receptors. String-like forms of vWf are typically found in the subendothelial matrix, versus plasma vWf, where vWf is bound to FVIII (not being able to change conformation readily), however newly released vWf from Weibel-palade bodies also unfurl while still attached to the surface of the endothelium (this is likely partly responsible for rolling of platelets on endothelial cells, although P-selectin-PSGL-1 interactions also mediate rolling).

Adhesion: In vessels of high shear rate (i.e. arteriolar microvasculature), vWf is essential for the first event in primary hemostasis – transient and weak platelet binding to the subendothelium to slow platelet velocity. vWf mediates platelet adhesion by acting as a bridge between the platelet glycoprotein Ib-IX-V receptor and subendothelial collagen or elastin microfibrils. Studies have shown that high molecular multimers of vWf are released from Weibel-Palade bodies after stimulation and unfold under shear as a “string”, still adherent to endothelial cells, acting like as a sling to “capture” circulating platelets (De Ceunynck et al 2013). Firm adhesion is mediated by integrin receptors binding to collagen, directly (GPIa/IIa) or indirectly by first binding to vWf (GPIIb/IIIa)
Platelet spreading: After adhesion, vWf facilitates platelet spreading on the subendothelial matrix by attaching to matrix proteins and the platelet integrin receptor GPIIb/IIIa on platelet surfaces.
Platelet aggregation: Von Willebrand factor aids platelet aggregation by acting as a bridge between GPIIb/IIIa on adjacent platelets, although fibrinogen is the main platelet aggregating agent in this regard. However, vWf may substitute for fibrinogen in afibrinogenemic patients.
Platelet plug stabilization: Von Willebrand factor may aid in stabilization of the primary platelet plug by aiding the incorporation of fibrin into the platelet plug. This is done by vWf by binding to GPIb-IX on platelets and onto fibrin.
Carrier for factor VIII: Von Willebrand factor acts as a carrier molecule for factor VIII, protecting it from degradation in the circulation and delivering it to sites of vessel injury. Thrombin breaks down the non-covalent association between the molecules, allowing factor VIII to participate in the coagulation cascade that takes place on platelet surfaces. In the absence of vWf, factor VIII coagulant (FVIII:C) activities in clotting assays are typically decreased (sometimes to very low levels, especially in human patients and less so in dogs [Stokol et al 1995]). In human patients, there is a variant of vWD, which is due to a genetic defect in the factor VIII-binding domain of vWf. The defective vWf cannot bind factor VIII, therefore it is rapidly cleared from the circulation, resulting in very low factor VIII:C activities, but normal vWf:Ag values, which mimics hemophilia A.
Inflammation: Studies from mice show that vWf may promote leukocyte recruitment (potentially indirectly via recruiting platelets). Leukocyte recruitment is reduced in vWf-knock out mice with some inflammation models, although this may be secondary to an overall defect in formation of Weibel-palade bodies in endothelial cells. vWf is also known to be a positive acute phase protein that is increased in the blood of human patients with inflammation (Kawecki et al review 2017).

Inheritance of vWD

Von Willebrand disease is inherited as an autosomal trait and is categorized into three types based on the amount and multimeric composition of the molecule.

Type I vWD: This is due to a deficiency in the amount of vWf. All multimers are present but in reduced amounts. This is the type reported in most breeds of dogs, including the Doberman. Bleeding is variable in this disorder and is dependent on the value of vWf:Ag and breed (as mentioned previously, Airedales have Type I vWD but rarely exhibit bleeding, in contrast to Dobermans, which also have type I vWD, but do bleed). In several breeds. the defect is thought to be due to a homozygous mutation in a splice site of the vWD gene.
Type II vWD: This has been reported in dogs (German Shorthair and Wirehair Pointers) (Brooks et al 1996, Kramer et al 2004) and in Quarterhorses and Thoroughbreds (Brooks et al 1991, Rathgeber et al 2001). It is characterized by low vWf:Ag and a relative decrease in high molecular weight multimers. The bleeding is more severe than in Type I vWD (Brooks et al 1996) and is not related to vWf:Ag (as vWf:Ag values do not correlate to multimers).
Type III vWD: This has been reported in dogs (Chesapeake Bay Retrievers, Scottish Terriers, Dutch Kooiker dogs and Shetland Sheepdogs), cats (Bieber et al 2014), pigs and a monkey (Raymond et al 1990, Patterson et al 2002, Nichols et al 2010). It is the most severe form of vWD and is characterized by an almost complete absence of vWf and very long buccal mucosal bleeding time. It is inherited as an autosomal recessive trait. In Dutch Kooiker (Slappendal et al 1998), the defect is due to a splice site mutation in intron 16 of the vWf gene, leading to production of a truncated transcript (Rieger et al 1996).

Clinical signs of vWD

Von Willebrand disease has been described in over 50 breeds of dogs. The trait is most prevalent in the Doberman Pinscher, Pembroke Welsh Corgi, Airedale Terrier, Scottish Terrier, and Shetland Sheepdog, but severely affected individuals or families have been identified in many purebred and mixed breeds (Brooks et al 1992). There are breed-differences in the proportion of carriers that actually express the vWD trait clinically through abnormal or excessive hemorrhage. e.g. Airedale Terriers rarely bleed, despite having a high prevalence of the trait, whereas Dobermans bleed quite commonly. The disease does occur in pigs, rabbits, cats and horses but has been recognized in only a few individuals of the latter two species. Severity of bleeding is highly variable in dogs affected with vWD.

In general, spontaneous bleeding tends to occur from mucous membranes lining the nose, mouth, urinary, reproductive, and intestinal tracts. Excessive bleeding in puppies may be noticed after tail docking, dewclaw removal, tattooing, or when the pup is teething. In less severely affected dogs, abnormal bleeding is seen only after surgery or trauma. Concurrent stress conditions such as viral and bacterial infections, hormonal fluctuations associated with heat cycles or pregnancy, and endocrine disorders causing deficiencies of steroid or thyroid hormones can all exacerbate signs of hemorrhage in dogs affected with vWD. Note that petechial hemorrhages are rarely seen in vWD, and if observed in a predisposed dog breed, a differential diagnosis of thrombocytopenia should precede vWD.

Diagnosis of vWD

Specific assays of canine vWf are needed to diagnose vWD in dogs as most dogs with vWD have normal platelet counts and coagulation profiles (PT, APTT, and fibrinogen). Puppies that show signs of bleeding but have normal platelet counts and coagulation test results should be tested for vWD, particularly if from a breed known to have the disease. The buccal mucosal bleeding time can be used as an in vivo test for vWD in the veterinary clinic (e.g. a Doberman with unknown vWf results prior to surgery). However, this test is not sensitive or specific for vWD, therefore it does not replace more specific vWf assays. There are a variety of laboratory assays for vWD, however the most commonly used assay is that for vWf:Ag measurement in a citrated plasma sample. In addition, there are now genetic tests for the splice site defects in specific brees, including type I vWD in Dobermans, Rottweilers and Airedales and type III vWD in Dutch Kooiker hounds and Scottish Terriers. The following intervals for plasma vWf:Ag have been established at the Comparative Coagulation Laboratory at Cornell University:

vWf:Ag result	Interpretation
70 to 180%	Normal
50 to 69%	Borderline
0 to 49%	vWD
<35%	At risk of hemorrhage

Normal: Dogs are considered clear of the vWD trait and are at low risk for expressing or transmitting the vWD trait.
Borderline: Dogs cannot be accurately classified as a carrier or clear of the trait on the basis of the vWf:Ag result. This is an overlap interval of plasma vWf:Ag, where some individuals are clear and some are carriers of vWD. On a second test, some borderline interval individuals fall in the normal or abnormal interval, thereby enabling a prediction of their genetic status. A test mating can be performed by breeding a borderline interval individual to a high-testing clear mate. If the borderline parent is clear of vWD, then all pups in the litter are predicted to be clear. The presence of one or more abnormal range pups indicates that the borderline parent is a carrier of vWD. Alternatively, these borderline dogs should be tested using a genetic test for vWD. This test is useful for confirming the carrier status in dogs with questionable vWf:Ag results, but should not be used to diagnose the disease.
Abnormal: Dogs are considered carriers of the vWD trait, and are at risk for transmitting an abnormal vWf gene to offspring. In some individuals this also represents a risk for expressing an abnormal bleeding tendency. In general, the lower the value of plasma vWf:Ag, the more at risk an individual dog is for expressing a bleeding tendency. Most dogs with bleeding due to vWD have vWf:Ag values < 35% (Brooks et al 1992, Stokol et al 1995).

Treatment of vWD

Plasma transfusion: Severe hemorrhage in vWD patients can be controlled with transfusion of fresh, fresh frozen plasma or cryoprecipitate. Whole blood should only be used in dogs that are hypoxic from anemia, and even then, component therapy (packed red blood cells and cryoprecipitate) is preferred as whole blood will not provide sufficient vWf to stop the hemorrhage. In addition, plasma products are optimal since these dogs usually require repeated transfusions through life and repeated exposure to red blood cell antigens increases the risk of transfusion reactions.
Desmopressin acetate (DDAVP): DDAVP stimulates the release of vWf from stores (Weibel-Palade bodies) in endothelial cells and increases vWf:Ag values and decreases the BMBT for up to 4 hours in Dobermans with type I vWD. Repeated administration has diminishing effectiveness due to depletion of stores. In addition, this drug does not work in dogs with type III vWD as they lack endothelial stores of vWf. The response to DDAVP in a single dog is repeatable and predictable but not all dogs with Type I vWD will respond to the drug, therefore it should not be relied upon to achieve hemostasis in surgery (but may useful as an adjunct to transfusion therapy). The dose of DDAVP is 1 µg/kg diluted in sterile saline given 30 minutes before surgery.