• The procoagulant nature of HIT can be simulated in a microfluidic model using human blood and its components.• PF4/glycosaminoglycans/ immunoglobulin G complexes activate monocytes through FcgRIIA to generate TF and thrombin, leading to coated platelets in HIT.Heparin-induced thrombocytopenia (HIT) is characterized by a high incidence of thrombosis, unlike other antibody-mediated causes of thrombocytopenia. We have shown that monocytes complexed with surface-bound platelet factor 4 (PF4) activated by HIT antibodies contribute to the prothrombotic state in vivo, but the mechanism by which this occurs and the relationship to the requirement for platelet activation via fragment crystallizable (Fc)gRIIA is uncertain. Using a microfluidic model and human or murine blood, we confirmed that activation of monocytes contributes to the prothrombotic state in HIT and showed that HIT antibodies bind to monocyte FcgRIIA, which activates spleen tyrosine kinase and leads to the generation of tissue factor (TF) and thrombin. The combination of direct platelet activation by HIT immune complexes through FcgRIIA and transactivation by monocyte-derived thrombin markedly increases Annexin V and factor Xa binding to platelets, consistent with the formation of procoagulant coated platelets. These data provide a model of HIT wherein a combination of direct FcgRIIA-mediated platelet activation and monocyte-derived thrombin contributes to thrombosis in HIT and identifies potential new targets for lessening this risk. (Blood. 2016;127(4):464-472) IntroductionHeparin-induced thrombocytopenia (HIT) is an iatrogenic, immunemediated disorder characterized by antibodies that recognize complexes between the platelet chemokine platelet factor 4 (PF4, CXCL4) and heparin or cell surface glycosaminoglycans (GAGs). 1,2 It is estimated that up to 50% of patients with HIT develop thrombosis that might be limb-and/or life-threatening. [3][4][5] Even with early recognition, cessation of heparin, and institution of alternative forms of anticoagulation, recurrent thromboembolic complications may occur and 10% to 20% of patients go on to amputation and/or death. 6 Thus, there is a need for a better understanding of the pathogenesis of HIT and to determine how this information can be used to mitigate the risk of thrombosis.Thrombocytopenia and thrombosis in HIT have been attributed to binding of PF4/heparin/immunoglobulin G (IgG) immune-complexes to the platelets through the IgG fragment crystallizable (Fc) region, which activates platelets through their immunoreceptor tyrosine-based activation motif (ITAM) receptor, FcgRIIA. 7,8 However, monocytes, endothelial cells, and other cell types might also be activated by these immune complexes and contribute to the underlying pathology, 9 but their contribution to the process is less well characterized. Indeed, recent evidence suggests that thrombosis in HIT is initiated by binding of pathogenic antibodies to antigenic complexes of PF4 and GAGs expressed by the endothelium as well as circulating cells, includi...

A prevailing dogma is that inhibition of vascular thrombosis by antagonizing platelet integrin αIIbβ3 cannot be achieved without compromising hemostasis, thus causing serious bleeding and increased morbidity and mortality. It is speculated that these adverse outcomes result from drug-induced activating conformational changes in αIIbβ3 but direct proof is lacking. Here, we report the structure-guided design of peptide Hr10 and a modified form of the partial agonist drug tirofiban that act as "pure" antagonists of αIIbβ3, i.e., they no longer induce the conformational changes in αIIbβ3. Both agents inhibit human platelet aggregation but preserve clot retraction. Hr10 and modified tirofiban are as effective as partial agonist drugs in inhibiting vascular thrombosis in humanized mice, but neither causes serious bleeding, establishing a causal link between partial agonism and impaired hemostasis. Pure orthosteric inhibitors of αIIbβ3 may thus provide safer alternatives for human therapy, and valuable tools to probe structure-activity relationships in integrins.

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Recently, we reported that human amniotic membrane-derived mesenchymal stem cells (AMMs) possess great angiogenic potential. In this study, we determined whether local injection of AMMs ameliorates peripheral neuropathy. AMMs were transplanted into injured sciatic nerves. AMM injection promoted significant recovery of motor nerve conduction velocity and voltage amplitude compared to human adipose-derived mesenchymal stem cells. AMM implantation also augmented blood perfusion and increased intraneural vascularity. Whole-mount fluorescent imaging analysis demonstrated that AMMs exhibited higher engraftment and endothelial incorporation abilities in the sciatic nerve. In addition, the higher expression of pro-angiogenic factors was detected in AMMs injected into the peripheral nerve. Therefore, these data provide novel therapeutic and mechanistic insights into stem cell biology, and AMM transplantation may represent an alternative therapeutic option for treating peripheral neuropathy.

B-domainless factor VIII (FVIII) ectopically expressed in megakaryocytes (MKs) is stored in α granules of platelets (pFVIII) and is capable of restoring hemostasis in FVIIInull mice, even in the presence of circulating inhibitors. However, our prior studies have shown that this ectopically expressed pFVIII can injure developing MKs. Moreover, the known risks of prolonged thrombocytopenia after bone marrow transplantation are significant challenges to the use of this strategy to treat individuals with severe hemophilia A and particularly those with intractable clinically relevant inhibitors. Because of these limitations, we now propose the alternative therapeutic pFVIII strategy of infusing pFVIII-expressing MKs or platelets derived from induced pluripotent stem cells (iPSCs). pFVIII-expressing iPSC-derived MKs, termed iMKs, release platelets that can contribute to improved hemostasis in problematic inhibitor patients with hemophilia A. As proof of principle, we demonstrate that hemostasis can be achieved in vitro and in vivo with pFVIII-expressing platelets and show prolonged efficacy. Notably, pFVIII-expressing platelets are also effective in the presence of inhibitors, and their effect was enhanced with recombinant FVIIa. Human pFVIII-expressing iMKs improved hemostasis in vitro, and derived platelets from infused human pFVIII-expressing iMKs improved hemostasis in FVIIInull mice. These studies indicate the potential therapeutic use of recurrent pFVIII-expressing MK or platelet infusions with prolonged hemostatic coverage that may be additive with bypassing agents in hemophilia A patients with neutralizing inhibitors.