In 2002 Brooks et al. (Blood 99:2434) described a colony of German shepherd dogs (GSD) with a hereditary bleeding diathesis. The affected GSD (aGSD) had normal fluid phase coagulation as well as normal platelet aggregation and secretion, but their platelets failed to generate prothrombinase activity and did not express phosphatidylserine (PS) upon activation with calcium ionophore. These features are characteristic of Scott Syndrome. In the current study, we defined a broader platelet function defect in aGSD, i.e. a failure to produce significant levels of coated-platelets. Coated-platelets, a subclass of activated platelets formed upon dual-agonist activation, express not only surface PS but also retain several alpha-granule proteins, support a robust prothrombinase activity and release microparticles (
J. Thromb. Haemost. 3:2185, 2005
). We activated platelets from control dogs and aGSD with convulxin plus thrombin and performed flow cytometric analyses to detect bound fibrinogen and membrane PS exposure. Post-stimulation, bound fibrinogen was detected on 34.6 ± 8.1 % of control dog platelets (mean ± 1SD; n=5) in contrast to 5.1 ± 2.5 % of aGSD platelets (n=5; p=0.006). In response to the same dual agonists, control dogs had 56.7 ± 10.3 % annexin-positive platelets while aGSD had only 4.7 ± 4.4 % (n=5; p=0.006). Control dogs and aGSD were also tested for their ability to release intracellular calcein upon dual agonist stimulation. As expected from studies with human platelets, control dog platelets released calcein from a sub-population of cells upon dual agonist stimulation; however, aGSD platelets demonstrated minimal loss of calcein [36.6 ± 6.4 % calcein negative control platelets (n=5); 3.5 ± 0.9 % calcein negative aGSD platelets (n=4; p=0.002)]. Additional activation events known to be associated with coated-platelet formation are currently being examined in aGSD to better define the basis of the platelet phenotype. Our results demonstrate that decreased production of coated-platelets is associated with abnormal bleeding and that a single hereditary defect is capable of abolishing prothrombinase activity and coated-platelet formation.
Disclosure: No relevant conflicts of interest to declare.
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