Moderate Pharmacological Platelet Count Reduction Limits Thrombus Formation without Hemostatic Impairment in Primates

Abstract Despite the established contribution of platelets to thrombotic cardiovascular disorders, documented in part by the effectiveness of platelet function inhibitors and the increased risk of thrombosis associated with high normal and supranormal platelet counts, relationships between circulating platelet count and thrombotic events remain largely undefined. Since the initiation and propagation of arterial, platelet-dependent thrombus must depend upon platelet count, albeit in a manner that could be nonlinear, we hypothesized that reducing platelet count within the normal range would produce an anti-thrombotic benefit with minimal effects on hemostasis. To test this hypothesis, we reduced the platelet count in baboons (n=4) by targeting the megakaryocyte growth and development factor thrombopoietin (TPO). A polyclonal anti-TPO autoantibody (anti-TPOab) was purified from the serum of a baboon that developed thrombocytopenia following recombinant TPO injections. The IC50 of the purified IgG fraction was found to be 0.76 μg/ml, determined using a proliferation assay with a TPO-dependent cell line. An i.v. bolus of the anti-TPO antiserum, 30–35 ml infused into baboons, resulted in a transient, >60% decrease in the circulating platelet count after 2–3 weeks. Other blood cell counts were unaffected vs. baseline values. The effect of platelet count reduction on thrombogenesis was evaluated using an established baboon arteriovenous (AV) shunt thrombosis model. Accumulation of 111-Indium-labeled platelets and 125-Iodine-labeled fibrinogen were measured within a 4 mm i.d. thrombogenic vascular graft segment that was deployed into a chronic AV shunt for 60 min. Blood flow was maintained at 100 ml/min, producing an arterial wall shear rate of 265 sec−1. Standard template bleeding times (BTs) were used to assess hemostatic impairment at various platelet counts. Platelet count reductions, ranging from 46–61% (normal levels averaging 352,000 ± 61,000 platelets/μl), reduced platelet deposition onto the graft surface by 46–68% (vs. control values of 4.1 ± 0.9 x 109 platelets deposited, n=9). Similarly, thrombus fibrin accumulation was reduced by 14–39% (vs. control values of 2.2 ± 0.4 mgs of deposited fibrin). Thrombus formation was not affected acutely by anti-TPOab administration, but correlated directly with circulating platelet numbers. As expected, BTs were not significantly prolonged until platelet counts fell below ~100,000 cells/μl. In contrast, single dose aspirin (32 mg/kg) at normal platelet counts did not significantly reduce graft associated platelet deposition in this model but doubled the BTs to 6.8 ± 2.6 min (vs. control values of 3.4 ± 0.9 min). With further reduction in platelet counts to 90,000 ± 30,000 platelets/μL, BTs were only slightly prolonged (5.6 ± 1.7 min, n=5). When platelet counts averaged 74,000 ± 20,000 platelets/μl in animals given ASA, BTs averaged only 9.4 ± 2.7 min (n=5). Thus ASA produced a hemostatic impairment that was approximately fixed (i.e., a BT prolongation of 3–4 min) and not disproportionately prolonged at reduced platelet counts. Thus specific lowering of the platelet count by pharmacologic inhibition of megakaryocytopoiesis may be an effective anti-thrombotic strategy in populations currently treated with conventional anti-platelet agents. Since direct inhibitors of platelet function produce a significant risk of bleeding, inhibition of platelet production may represent a safer approach for reducing the pro-thrombotic capacity of the circulating platelet pool.