ALBERT

Description: Abstract 3326 Immune thrombocytopenia (ITP) is a relatively prevalent disease in dogs with significant morbidity and mortality. Canine ITP is clinically analogous to human ITP, with heterogeneity in bleeding manifestations in individuals with similar platelet counts. With a view to ultimately investigate this bleeding heterogeneity, we set out to develop a canine model of ITP. There are currently no existing large animal models of ITP. An induced canine ITP model would be representative of ITP without the confounding co-morbidities seen in clinical cases. Since spontaneous ITP occurs in both dogs and humans, the dog is an ideal translational model. We hypothesized that 2F9, a murine IgG2a monoclonal antibody to the canine platelet glycoprotein GPIIb (a common target of autoantibodies in ITP), would induce predictable dose-dependent thrombocytopenia (TCP) in healthy dogs. 2F9 had not been previously administered in vivo. We produced highly purified 2F9 and αYFA antibodies from the 2F9 hybridoma (gift of David Wilcox, Blood Research Institute, Wisconsin) and an isotype control murine anti-yellow fever antibody (αYFA) hybridoma. A dose titration (2 dogs) and a dose repeatability study (3 dogs) were performed in healthy adult research dogs by repeated intravenous infusion (≤ 6 doses) of 2F9 antibody until a target nadir of 5–30 × 103 platelets/μl was reached. Platelet counts were performed hourly until the platelet count reached the desired nadir range (t=0 hrs), after which complete blood counts were performed at 2, 4, 6, 8, 12, 24 hours, then q 24 hours for 10 days. The following were evaluated throughout the study: physical examination, buccal mucosal bleeding time (BMBT, baseline and t=0 only), serum cytokines and chemokines (INFγ, Interleukin (IL) 2, 6, 7, 8, 10, 15, 18, KC, IP-10, MCP-1, GM-CSF, TNFα; Milliplex CCYTOMAG-90K), fibrinogen, and D-dimers. Specificity of the 2F9 effect was confirmed by IV infusion of the isotype control (αYFA) to 3 dogs at the highest cumulative effective dose of 2F9 (167 μg/kg); all parameters were measured as above (t=0 hrs was one hour after αYFA dosing). Within 2 hours of a median cumulative 2F9 administration of 63 μg/kg (range 50.0–166.6 μg/kg), all dogs developed profound TCP (range 11–28 × 103/μl). Compared to the control group, platelet nadir was significantly lower (median (range): 6 (4–11) × 103/μl vs. 200 (179–209) × 103/μl; p= 0.036) and change in platelet count from baseline to nadir was significantly greater in the 2F9-treated group (median (range): 238 (179–325) × 103/μl vs. 4 (0–10) × 103/μl; p=0.036) (Fig 1); p-values were calculated using the exact Wilcoxon rank-sum test. Platelet nadir was in our target range and platelet count remained 〈 40 × 103/μl in all 2F9-treated dogs for 24 hours. Dosing was predictable: in each dog, after an initial dose of 50 μg/kg 2F9, the second dose needed to reach the target nadir could be accurately calculated from the initial platelet decrease. 2F9-treated dogs developed a range of clinical bleeding from none to petechiae, ecchymoses, melena, and hematuria. At t=0 hrs, BMBT increased 3–8 fold in treated dogs, compared to 〈 2 fold in control dogs. Dogs had no changes in vital signs or demeanor and did not require any transfusion support. The model does not appear pro-thrombotic as fibrinogen and D-dimers were similar over time in 2F9-treated vs. control dogs. 2F9 infusion also generated negligible systemic inflammation, as assessed by white blood cell count and serum cytokine measurement. Unexpectedly, however, serum IL8 tracked faithfully with platelet count, demonstrating that platelets are a major source of serum IL8 in dogs (Fig 2). Although α granules are known to contain IL8, platelets have not been previously described as a significant serum IL8 source. Since IL8 is an important neutrophil chemokine, our finding may illuminate a novel mechanism of platelet-neutrophil cross-talk. In summary, we have developed a novel large animal ITP model that is highly representative of the spontaneous disease. Like naturally-occurring ITP, dogs demonstrate bleeding heterogeneity despite similar platelet counts (data not shown). We expect our model to lead to further insights into bleeding mechanisms in ITP. Ultimately, understanding what factors predispose certain patients to bleed will allow us to exploit these factors therapeutically as novel ITP treatments. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2193 In both canine and human patients with Immune Thrombocytopenia (ITP), bleeding risk is challenging to predict, and potentially leads to over-treatment of patients at low risk. Conversely, recent studies have highlighted the risk of thrombosis in ITP during platelet recovery. Given these clinical observations, we hypothesized that in ITP, changes in platelet response to agonists may occur in addition to changes in platelet numbers. In response to dual agonist activation (thrombin and convulxin), a subpopulation of platelets in both humans and dogs develops enhanced procoagulant activity. This subpopulation is termed coated platelets, and differences in individuals' potential to form coated platelets have been correlated with both hemorrhagic and thrombotic outcomes. In this exploratory study, we serially evaluated ex vivo platelet responsiveness to both thrombin and dual agonists (termed coated platelet potential) in a novel canine model of ITP. Dogs (n=4) were infused with a murine monoclonal anti-GPIIb antibody (2F9) in order to model ITP and generate predictable severe thrombocytopenia. Control dogs (n=3) were infused with a control antibody. Platelet count, thrombin responsiveness, and coated platelet potential were measured at baseline, time zero, 6 hours, 24 hours, and every 24hrs thereafter until the platelet count was ≥ baseline for at least two consecutive measures (recovery). Time zero was defined as the time when platelet count first fell to ≤ 30,000/μl following 2F9 infusion, or 1 hour following control antibody infusion. For platelet thrombin responsiveness, a monoclonal antibody to P-selectin was used to determine platelet P-selectin surface expression by flow cytometry after stimulation with graded doses of thrombin. The ED50 Thrombin was defined as the concentration of thrombin required for half-maximal P-selectin expression. Coated platelet potential was defined as the percent of platelets activated to the highly procoagulant state after dual stimulation with thrombin and convulxin, as determined by binding of biotinylated fibrinogen by platelets by flow cytometry. All dogs in the treated group developed severe thrombocytopenia (median=6×103, range=4–11×103 platelets/uL); no dogs in the control group developed thrombocytopenia. All treated dogs had platelet recovery by 240 hours (median=132 hours, range 120–240hours). Of interest, at 6 hours, ED50 Thrombin in the treated group increased nearly twofold (fig 1A) (ratio of median ED50 Thrombin treated/baseline=1.6, range 1.3–2.3), which correlated with a decline in coated platelet potential by nearly half of baseline (fig 1B) (median 52.4% of baseline, range 19.6–61.5%); minimal change from baseline was observed in controls. In both groups, ED50 Thrombin was lower at recovery than baseline (fig 1A) (treated median ED50 Thrombin=71.5% of baseline; control median ED50 Thrombin=67% of baseline). A trend of rising coated platelet potential was also noted as platelets recovered in the treated group. In conclusion, in this exploratory study of a canine model of ITP, we observed dynamic changes in platelet responsiveness. During severe thrombocytopenia, we observed a rise in ED50, indicating a decline in response to thrombin, which correlated with a fall in coated platelet potential. We speculate that this early fall in platelet thrombin response and coated platelet potential could contribute to hemorrhage risk in ITP. As a complement to this finding, in the treated group, there was a rise in coated platelet potential as platelets rebounded and coated platelet potential was slightly greater than baseline at recovery. This is consistent with others' observation that younger platelets are more likely to have coated platelet potential. We also observed a decline in ED50 Thrombin at recovery, not only in the treated dogs, but also control dogs. Thus, at recovery, the decline in ED50 Thrombin was independent of treatment group. However, this may be an artifact of our small sample size. Our observed increase in coated platelet potential during platelet recovery could potentially contribute to the thrombotic tendency of some ITP patients. Future studies are planned to explore the relationship of hemorrhagic and thrombotic risk with platelet thrombin responsiveness and coated platelet potential in this model of ITP and clinical studies of canine and human ITP. Disclosures: No relevant conflicts of interest to declare.

Description: The anti-bleeding therapy recombinant factor VIIa (rFVIIa) is used to abrogate bleeding in hemophiliacs with inhibitors, bypassing the need for replacement factors. RFVIIa is hypothesized to work by increasing Xa generation on the platelet’s surface. However, high plasma levels of rFVIIa are required, in part due to the weak binding of rFVIIa to platelets. We hypothesized that the efficacy of the therapy could be improved by administering rFVIIa already bound to platelets. One platelet preparative that may be used in this application is rehydrated, lyophilized (RL) platelets. RL platelets are fixed with paraformaldehyde, which allows them to be frozen and lyophilized while retaining their hemostatic capabilities. Previously, we have shown RL platelets are capable of supporting rFVIIa-mediated thrombin generation and that thrombin generation is increased in a rFVIIa dose-dependent manner (Blood, 106:4057, 2005). In this current study, we have characterized the ability of RL platelets to modulate rFVIIa-mediated thrombin generation and fibrin clot formation in a cell-based complete model of hemophilia. The addition of RL platelets with 50 nM rFVIIa increased the thrombin generation rate in hemophilia 2.8-fold more than 50 nM rFVIIa, alone. Further, the addition of RL platelets with 50 nM rFVIIa normalized clot formation and stability in a fibrinolytic environment, which did not occur in the presence of rFVIIa, alone. In contrast, the addition of RL platelets, alone, to hemophilic conditions had minimal to no effect on thrombin generation rate or the onset of clot formation, suggesting that the effects were due to a specific interaction between rFVIIa and RL platelets. When rFVIIa plus RL platelets were added to platelet-rich plasma from patients with hemophilia A in the presence of tissue-type plasminogen activator, clot formation and stability were improved more than the addition of either agent alone. To examine the mechanism of RL platelets’ augmentation of rFVIIa activity, we titrated the phosphatidylserine (PS) binding protein, annexin V, into reactions with RL platelets in the presence of factors Xa, Va, and prothrombin and measured thrombin generation. The addition of annexin V reduced thrombin generation equally in reactions that contained RL platelets stimulated with or without SFLLRN. Further, thrombin generation was similar on RL platelets simulated with or without SFLLRN in the absence of annexin V. These data suggest that RL platelets already have PS exposed on their surface. We conclude that RL platelets can support rFVIIa-mediated thrombin generation in the absence of factor IX and may enhance rFVIIa activity in part due to PS exposure on the RL platelet surface. We hypothesize that co-administration of RL platelets with rFVIIa may increase the efficacy of rFVIIa, at lower doses of rFVIIa than are currently required to achieve hemostasis.

Description: The anti-bleeding therapy, recombinant factor VIIa (rFVIIa), is thought to bind to the platelet’s surface and increase thrombin generation in hemophilia. However, high plasma levels of rFVIIa are required, in part, due to the weak binding of rFVIIa to platelets. We hypothesized that the efficacy of the therapy could be improved by administering rFVIIa already bound to platelets. A recently described protocol involving pretreatment of platelets with paraformaldehyde permits platelets to be lyophilized while preserving many platelet functions. Such platelets could be used for binding rFVIIa ex vivo and then administered to hemophilic patients during a bleeding event. In this study, we have characterized the ability of reconstituted, lyophilized (RL) platelets to support thrombin generation under normal and hemophilic conditions and in the presence of rFVIIa. First, freshly-isolated (control) or RL platelets were incubated with factors IXa, VIII(a), X, V and II in the presence of 3 mM CaCl2 and assayed for thrombin generation. In these assays, both freshly-isolated and RL platelets supported thrombin generation (1.15x10−4 +/− 5.37x10−5 mOD/min2/platelet and 8.46x10−3 +/− 4.78x10−3 mOD/min2/platelet, respectively). In the absence of factor IX (hemophilia B), thrombin generation was significantly reduced on both freshly-isolated and RL platelets (4.19x10−6 +/− 4.50x10−6 mOD/min2/platelet and 8.25x10−4 +/− 1.13x10−6 mOD/min2/platelet, respectively). Interestingly, RL platelets supported 10 – 100-fold higher thrombin generation rates than fresh thrombin-activated platelets. Second, we examined the activity of rFVIIa on RL platelets in the absence of factors IX and VIII. RFVIIa increased thrombin generation on RL platelets in a rFVIIa-concentration dependent manner (between 1nM and 150nM), similar to that seen when using fresh platelets. An inhibitory anti-tissue factor (TF) antibody did not affect rFVIIa-mediated thrombin generation on RL platelets, indicating that the activity of rFVIIa on RL platelets is independent of TF. Finally, we examined the effect of different platelet agonists (thrombin, convulxin, and A23187) on fresh and RL platelets. When fresh platelets are stimulated with A23187 or co-stimulated with thrombin and convulxin, they become more procoagulant than platelets activated with thrombin alone. However, stimulation of RL platelets with A23187 or co-stimulation with thrombin and convulxin did not increase thrombin generation versus thrombin alone. RL platelets stimulated with thrombin, alone, had 3.1-fold higher activity than thrombin- and convulxin-costimulated fresh platelets, but 1.4-fold lower activity than A23187-stimulated fresh platelets. These data suggest that RL platelets are in a maximally active state prior to the addition of platelet agonists. We conclude that RL platelets are procoagulant and can support rFVIIa-mediated thrombin generation in the absence of factor IX. We hypothesize that co-administration of RL platelets with rFVIIa may increase the efficacy of rFVIIa treatment.