Formation of Procoagulant Platelets in Heparin-Induced Thrombocytopenia (HIT) Follows a Unique Signaling Pathway

Abstract 197

HIT is a drug-induced, immune-mediated thrombocytopenia and thrombosis disorder associated with considerable morbidity and mortality. HIT is associated with generation of procoagulant platelet microparticles (Warkentin et al., Blood 1994; 84:3691), one of the cardinal features of procoagulant platelets, along with a phosphatidylserine-positive (PS+) external plasma membrane and surface retention of procoagulant proteins from plasma or released from platelet γ-granules. Other names for procoagulant platelets include COAT, or coated, platelets (Dale, J Thromb Haemostas 2005; 3:2185). To date, virtually all of the work on procoagulant platelets has used collagen or convulxin signaling via ITAM-associated GPVI, combined with thrombin signaling via GPCRs PAR1/PAR4. Batar and Dale reported the formation of coated platelets as a result of dual platelet FcγRIIa and thrombin stimulation; they used crosslinking of an anti-FcγRIIa antibody or anti-platelet glycoprotein antibodies which also engaged FcγRIIa (J Lab Clin Med 2001; 138:393). We report here the procoagulant activation of human and FcγRIIa transgenic (tg) mouse platelets stimulated by the HIT immune complex (IC) and thrombin (thr). HIT IC consist of ultralarge complexes of heparin and platelet factor 4 (hep/PF4) bound by HIT-like monoclonal antibody KKO. Washed human platelets, wild-type mouse platelets, or FcγRIIa tg mouse platelets were stimulated with the HIT IC + thr. Controls include convulxin (100 ng/ml) + thr, HIT IC alone, or thr alone as the stimulus. Human platelets and FcγRIIa-tg platelets, but not wild-type mouse platelets, develop a 40 to 60% PS+ population in response to HIT IC (100 ug/ml KKO)+ thr (0.5 U/ml), but not to either alone, as assessed by flow cytometry using labeled Annexin V. Unlike cvx + thr, which consistently shows a broad distribution of PS+ platelets in our hands and in the literature, HIT IC-stimulated platelets reproducibly show one discrete PS+ subpopulation (n = 6 per group). Preincubation of platelets with novel specific Syk inhibitor PRT318 (Reilly et al., Blood 2011; 117:2241) completely prevented PS+ platelet formation. FcγRIIa-tg platelets null for Akt2 showed a ∼50% reduction in the formation of PS+ platelets. Studies with FcγRIIa-tg mice null for Cal-DAG GEFI are ongoing. Understanding the unique pattern of procoagulant platelets generated by HIT IC + thr and the signals which produce it may lead to new diagnostic tests and therapeutic interventions.