Characterization of a Heritable Bleeding Disorder in a Horse: Reduce PS-Flip Leads to Progressive Dysfunction in the Common Pathway of Platelet Coagulation.
Authors: Norris Jeffrey W. , Auh Joong-Hyuck , Wilson Sandra , Clutter Dana S. , Crowe John H. , Tablin Fern 
Published: October 2019

Abstract
A 3 year old Thoroughbred horse presented to the Veterinary Teaching Hospital with a three week history of bleeding. Clinical laboratory findings showed normal complete blood count including a normal platelet count. In addition the horse had normal prothrombin and partial thromboplastin times, as well as normal coagulation factors. These data suggested a defect in platelet function. The common pathway of the coagulation cascade occurs at the platelet surface when phosphatidylserine (PS) is flipped to the outer leaflet of the platelet membrane, providing binding sites for the prothrombinase complex that catalyzes the production of thrombin, which carries out the proteolytic activation of fibrinogen. We characterized the capacity of horse’s platelets to carry out each of these steps. PS-flip in the patient’s platelets was induced using several physiological agonists and found to be significantly reduced after stimulation with thrombin (0.1U/ml). The PS was detected by labeling platelets with FITC-annexin V, and measuring FITC intensity by flow cytometry. In comparison to control platelets, approximately 50% of the patient’s platelets were positive for FITC-annexin V after stimulation with thrombin. The fluorescent intensity of the label on the patient’s positive platelets was 40% that of controls. The reduced PS on the patient’s platelets represents a potential loss of prothrombinase binding sites. Using a prothombinase assay based on the hydrolysis the chromogenic substrate S-2238, we determined the amount of thrombin produced by the patient’s platelets over time. In response to thrombin (0.1U/ml), the patient’s platelets produced thrombin at a rate of 0.023±0.002 NIH-U/min/1 X 108 platelets, which was significantly less than control platelets, 0.053±0.004 NIH-U/min/1 X 108 platelets. Neither resting platelets nor the thrombin used to stimulate them produced appreciable hydrolysis of S-2238. We correlated this reduced prothrombinase activity with the capacity of the patient’s platelets bind FITC-fibrinogen. After stimulation with thrombin (0.1U/ml), control platelets form platelet microaggregates that are intensely labeled with FITC-fibrinogen and can be observed by flow cytometry. The patient’s platelets do not form platelet microaggregates and the FITC-fibrinogen labeling is only 20% that of control platelets. This reduced FITC-fibrinogen labeling is not due to a defect in the function of the a2bb3a integrin, because the patient’s platelets bind normal amounts of FITC-fibrinogen in response to ADP. In this regard, the affected horse is unlike patients with Glannzmann’s thrombasthenia, who cannot bind fibrinogen. Instead, the dysfunction is more closely related to individuals with Scott syndrome, who cannot produce the PS surface that is required for assembly of the prothrombinase and ultimately activation of fibrinogen. Ongoing studies using the techniques described above demonstrate one affected and one normal progeny from the original patient.