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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., Current inhibitors of platelet integrin αIIbβ3 cause excessive bleeding, which limited their clinical use in cardiac patients. Here the authors design pure orthosteric αIIbβ3 inhibitors that prevent platelet aggregation and thrombosis without causing bleeding in humanized mouse models of thrombosis

Cadherins play a major role in mediating cell–cell adhesion, which shares many parallels with platelet–platelet interactions during aggregate formation and clot stabilization. Platelets express epithelial (E)-cadherin, but its contribution to platelet function and/or platelet production is currently unknown. To assess the role of E-cadherin in platelet production and function in vitro and in vivo, we utilized a megakaryocyte-specific E-cadherin knockout mouse model. Loss of E-cadherin in megakaryocytes does not affect megakaryocyte maturation, platelet number or size. However, platelet dysfunction in the absence of E-cadherin is revealed when conditional knockout mice are challenged with acute antibody-mediated platelet depletion. Unlike wild-type mice that recover fully, knockout mice die within 72 hours post-antibody administration, likely from haemorrhage. Furthermore, conditional knockout mice have prolonged tail bleeding times, unstable clot formation, reduced clot retraction and reduced fibrin deposition in in vivo injury models. Murine platelet aggregation in vitro in response to thrombin and thrombin receptor activating peptide is compromised in E-cadherin null platelets, while aggregation in response to adenosine diphosphate (ADP) is not significantly different. Consistent with this, in vitro aggregation of primary human platelets in response to thrombin is decreased by an inhibitory E-cadherin antibody. Integrin activation and granule secretion in response to ADP and thrombin are not affected in E-cadherin null platelets, but Akt and glycogen synthase kinase 3β (GSK3β) activation are attenuated, suggesting a that E-cadherin contributes to aggregation, clot stabilization and retraction that is mediated by phosphoinositide 3-kinase/Akt/GSK3β signalling. In summary, E-cadherin plays a salient role in platelet aggregation and clot stability.

Heparin-induced thrombocytopenia (HIT) is a prothrombotic disorder initiated by antibodies to platelet factor 4 (PF4)/heparin complexes. PF4 released from platelets binds to surface glycosaminoglycans on hematopoietic and vascular cells that are heterogenous in composition and differ in affinity for PF4. PF4 binds to monocytes with higher affinity than to platelets, and depletion of monocytes exacerbates thrombocytopenia in a murine HIT model. Here we show that the expression of PF4 on platelets and development of thrombocytopenia are modulated by the (re)distribution of PF4 among hematopoietic and endothelial cell surfaces. Binding of PF4 to platelets in whole blood in vitro varies inversely with the white cell count, likely because of the greater affinity of monocytes for PF4. In mice, monocyte depletion increased binding of PF4 to platelets by two- to three-fold. Induction of HIT in mice caused a transient >80-fold increase in binding of HIT antibody to monocytes vs 3.5-fold increase to platelets and rapid transient monocytopenia. Normalization of monocyte counts preceded the return in platelet counts. Exposure of blood to endothelial cells also depletes PF4 from platelet surfaces. These studies demonstrate a dynamic interchange of surface-bound PF4 among hematopoetic and vascular cells that may limit thrombocytopenia at the expense of promoting prothrombotic processes in HIT.

In vitro-grown megakaryocytes for generating platelets may have value in meeting the increasing demand for platelet transfusions. Remaining challenges have included the poor yield and quality of in vitro-generated platelets. We have shown that infusing megakaryocytes leads to intrapulmonary release of functional platelets. A Src kinase inhibitor (SU6656), a Rho-associated kinase inhibitor (Y27632), and an aurora B kinase inhibitor (AZD1152) have been shown to increase megakaryocyte ploidy and in vitro proplatelet release. We now tested whether megakaryocytes generated from CD34+ hematopoietic cells in the presence of these inhibitors could enhance functional platelet yield following megakaryocyte infusion. As expected, all inhibitors increased megakaryocyte ploidy, size, and granularity, but these inhibitors differed in whether they injured terminal megakaryocytes: SU6656 was protective, whereas Y27632 and AZD1152 increased injury. Upon infusion, inhibitor-treated megakaryocytes released threefold to ninefold more platelets per initial noninjured megakaryocyte relative to control, but only SU6656-treated megakaryocytes had a significant increase in platelet yield when calculated based on the number of initial CD34+ cells; this was fourfold over nontreated megakaryocytes. The released platelets from drug-treated, but healthy, megakaryocytes contained similar percentages of young, uninjured platelets that robustly responded to agonists and were well incorporated into a growing thrombus in vivo as controls. These studies suggest that drug screens that select megakaryocytes with enhanced ploidy, cell size, and granularity may include a subset of drugs that can enhance the yield and function of platelets, and may have clinical application for ex vivo-generated megakaryocytes and platelet transfusion.

Stem cell-derived platelets have the potential to replace donor platelets for transfusion. Defining the platelet-producing megakaryocytes (MKs) within the heterogeneous MK culture may help to optimize the in vitro generation of platelets. Using 2 human stem cell models of megakaryopoiesis, we identified novel MK populations corresponding to distinct maturation stages. An immature, low granular (LG) MK pool (defined by side scatter on flow cytometry) gives rise to a mature high granular (HG) pool, which then becomes damaged by apoptosis and glycoprotein Ib α chain (CD42b) shedding. We define an undamaged HG/CD42b+ MK subpopulation, which endocytoses fluorescently labeled coagulation factor V (FV) from the media into α-granules and releases functional FV+CD42b+ human platelet-like particles in vitro and when infused into immunodeficient mice. Importantly, these FV+ particles have the same size distribution as infused human donor platelets and are preferentially incorporated into clots after laser injury. Using drugs to protect HG MKs from apoptosis and CD42b shedding, we also demonstrate that apoptosis precedes CD42b shedding and that apoptosis inhibition enriches the FV+ HG/CD42b+ MKs, leading to increased platelet yield in vivo, but not in vitro. These studies identify a transition between distinct MK populations in vitro, including one that is primed for platelet release. Technologies to optimize and select these platelet-ready MKs may be important to efficiently generate functional platelets from in vitro-grown MKs.

Friend leukemia virus integration 1 (FLI1), a critical transcription factor (TF) during megakaryocyte differentiation, is among genes hemizygously deleted in Jacobsen syndrome, resulting in a macrothrombocytopenia termed Paris-Trousseau syndrome (PTSx). Recently, heterozygote human FLI1 mutations have been ascribed to cause thrombocytopenia. We studied induced-pluripotent stem cell (iPSC)-derived megakaryocytes (iMegs) to better understand these clinical disorders, beginning with iPSCs generated from a patient with PTSx and iPSCs from a control line with a targeted heterozygous FLI1 knockout (FLI1+/-). PTSx and FLI1+/- iMegs replicate many of the described megakaryocyte/platelet features, including a decrease in iMeg yield and fewer platelets released per iMeg. Platelets released in vivo from infusion of these iMegs had poor half-lives and functionality. We noted that the closely linked E26 transformation-specific proto-oncogene 1 (ETS1) is overexpressed in these FLI1-deficient iMegs, suggesting FLI1 negatively regulates ETS1 in megakaryopoiesis. Finally, we examined whether FLI1 overexpression would affect megakaryopoiesis and thrombopoiesis. We found increased yield of noninjured, in vitro iMeg yield and increased in vivo yield, half-life, and functionality of released platelets. These studies confirm FLI1 heterozygosity results in pleiotropic defects similar to those noted with other critical megakaryocyte-specific TFs; however, unlike those TFs, FLI1 overexpression improved yield and functionality.

Heparin-induced thrombocytopenia (HIT) is a prothrombotic disorder initiated by antibodies against complexes between human platelet factor 4 (hPF4) and heparin. A better understanding of the events that initiate the prothrombotic state may improve approaches to antithrombotic management. Here, we visualized thrombus formation in an in vivo murine model and an endothelialized microfluidic system that simulate the pathogenesis of HIT. hPF4 released from platelets predominantly bound to peri-injury endothelium and formed HIT antigenic complexes that were dissociated by heparin. In mice expressing both hPF4+ and human platelet IgG Fc receptor IIA (FcγRIIA), infusion of the HIT-like monoclonal antibody KKO increased fibrin and platelet deposition at sites of injury, followed immediately by antigen formation on proximate endothelial cells. After a few minutes, HIT antigen was detected within the thrombus itself at the interface between the platelet core and the surrounding shell. We observed similar results in the humanized, endothelialized microfluidic system. hPF4 and KKO selectively bound to photochemically injured endothelium at sites where surface glycocalyx was reduced. These studies support the concept that the perithrombus endothelium is the predominant site of HIT antigen assembly. This suggests that disrupting antigen formation along the endothelium or protecting the endothelium may provide a therapeutic opportunity to prevent thrombotic complications of HIT, while sparing systemic hemostatic pathways.

A prevailing dogma is that effective inhibition of vascular thrombosis by therapeutic targeting of platelet integrin αIIbβ3 cannot be achieved without compromising hemostasis. The resulting serious bleeding and increased morbidity and mortality have limited use of current anti-αIIbβ3 drugs to high-risk cardiac patients. It is speculated that these adverse outcomes result from drug-induced conformational changes in αIIbβ3 but direct proof is lacking. We used structure-guided design to generate the ligand-mimetic peptide Hr10 and a modified form of the partial agonist drug Tirofiban that now act as “pure” orthosteric antagonists of αIIbβ3, i.e. they no longer induce the conformational changes in αIIbβ3 and also suppress these changes in presence of agonists. Both agents inhibited human platelet aggregation effectively but without interfering with clot retraction. When tested in a humanized mouse model of thrombosis predictive of clinical efficacy, Hr10 was as effective as the αIIbβ3 partial agonist peptide drug Eptifibatide in inhibiting arteriolar thrombosis, but in sharp contrast to Eptifibatide, Hr10 did not cause serious bleeding, establishing a causal link between partial agonism and impaired hemostasis. Pure orthosteric inhibitors of αIIbβ3 may thus offer safer alternatives for human therapy. Our structure-guided approach may also find utility in designing similar drug candidates targeting other integrins and in providing vital tools for further probing structure-activity relationships in integrins.

BACKGROUND Acquired thrombotic thrombocytopenic purpura (TTP) is a potentially fatal disease in which ultralarge von Willebrand factor (UL-VWF) multimers accumulate as a result of autoantibody inhibition of the VWF protease, ADAMTS13. Current treatment is not specifically directed at the responsible autoantibodies and in some cases is ineffective or of transient benefit. More rational, reliable, and durable therapies are needed, and a human autoantibody-mediated animal model would be useful for their development. Previously, TTP patient anti-ADAMTS13 single-chain variable-region fragments (scFv's) were cloned that inhibited ADAMTS13 proteolytic activity in vitro and expressed features in common with inhibitory immunoglobulin G in patient plasma. Here, pathogenicity of these scFv's is explored in vivo by transfecting mice with inhibitory antibody cDNA. STUDY DESIGN AND METHODS Hydrodynamic tail vein injection of naked DNA encoding human anti-ADAMTS13 scFv was used to create sustained ADAMTS13 inhibition in mice. Accumulation of UL-VWF multimers was measured and formation of platelet (PLT) thrombi after focal or systemic vascular injury was examined. RESULTS Transfected mice expressed physiological plasma levels of human scFv and developed sustained ADAMTS13 inhibition and accumulation of unprocessed UL-VWF multimers. Induced focal endothelial injury generated PLT thrombi extending well beyond the site of initial injury, and systemic endothelial injury induced thrombocytopenia, schistocyte formation, PLT thrombi, and death. CONCLUSIONS These results demonstrate for the first time the ability of human recombinant monovalent anti-ADAMTS13 antibody fragments to recapitulate key pathologic features of untreated acquired TTP in vivo, validating their clinical significance and providing an animal model for testing novel targeted therapeutic approaches.

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)γRIIA 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 FcγRIIA, 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 FcγRIIA 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 FcγRIIA-mediated platelet activation and monocyte-derived thrombin contributes to thrombosis in HIT and identifies potential new targets for lessening this risk.

The use of fibrinolytic agents to prevent new thrombus formation is limited by an increased risk of bleeding due to lysis of hemostatic clots that prevent hemorrhage in damaged blood vessels. We sought to develop an agent that provides thromboprophylaxis without carrying a significant risk of causing systemic fibrinolysis or disrupting hemostatic clots. We previously showed that platelet (PLT) α granule-delivered urokinase plasminogen activator (uPA) is highly effective in preventing thrombosis, while being associated with little systemic fibrinolysis or bleeding. Here, we generated a chimeric prodrug composed of a single-chain version of the variable region of an anti-αIIbβ3 mAb fused to a thrombin-activatable, low-molecular-weight pro-uPA (PLT/uPA-T). PLT/uPA-T recognizes human αIIbβ3 on both quiescent and activated platelets and is enzymatically activated specifically by thrombin. We found that this prodrug binds tightly to human platelets even after gel filtration, has a prolonged half-life in mice transgenic for human αIIb compared with that of uPA-T, and prevents clot formation in a microfluidic system. Importantly, in two murine injury models, PLT/uPA-T did not lyse preexisting clots, even when administration was delayed by as little as 10 minutes, while it concurrently prevented the development of nascent thrombi. Thus, PLT/uPA-T represents the prototype of a platelet-targeted thromboprophylactic agent that selectively targets nascent over preexisting thrombi.

Protein disulfide isomerase (PDI) has two distinct CGHC redox-active sites; however, the contribution of these sites during different physiologic reactions, including thrombosis, is unknown. Here, we evaluated the role of PDI and redox-active sites of PDI in thrombosis by generating mice with blood cells and vessel wall cells lacking PDI (Mx1-Cre Pdifl/fl mice) and transgenic mice harboring PDI that lacks a functional C-terminal CGHC motif [PDI(ss-oo) mice]. Both mouse models showed decreased fibrin deposition and platelet accumulation in laser-induced cremaster arteriole injury, and PDI(ss-oo) mice had attenuated platelet accumulation in FeCl3-induced mesenteric arterial injury. These defects were rescued by infusion of recombinant PDI containing only a functional C-terminal CGHC motif [PDI(oo-ss)]. PDI infusion restored fibrin formation, but not platelet accumulation, in eptifibatide-treated wild-type mice, suggesting a direct role of PDI in coagulation. In vitro aggregation of platelets from PDI(ss-oo) mice and PDI-null platelets was reduced; however, this defect was rescued by recombinant PDI(oo-ss). In human platelets, recombinant PDI(ss-oo) inhibited aggregation, while recombinant PDI(oo-ss) potentiated aggregation. Platelet secretion assays demonstrated that the C-terminal CGHC motif of PDI is important for P-selectin expression and ATP secretion through a non-αIIbβ3 substrate. In summary, our results indicate that the C-terminal CGHC motif of PDI is important for platelet function and coagulation.

Heparin-induced thrombocytopenia (HIT) is a prothrombotic disorder mediated by complexes between platelet factor 4 (PF4) and heparin or other polyanions, but the risk of thrombosis extends beyond exposure to heparin implicating other PF4 partners. We recently reported that peri-thrombus endothelium is targeted by HIT antibodies, but the binding site(s) has not been identified. We now show that PF4 binds at multiple discrete sites along the surface of extended strings of von Willebrand factor (VWF) released from the endothelium following photochemical injury in an endothelialized microfluidic system under flow. The HIT-like monoclonal antibody KKO and HIT patient antibodies recognize PF4-VWF complexes, promoting platelet adhesion and enlargement of thrombi within the microfluidic channels. Platelet adhesion to the PF4-VWF-HIT antibody complexes is inhibited by antibodies that block FcγRIIA or the glycoprotein Ib-IX complex on platelets. Disruption of PF4-VWF-HIT antibody complexes by drugs that prevent or block VWF oligomerization attenuate thrombus formation in a murine model of HIT. Together, these studies demonstrate assembly of HIT immune complexes along VWF strings released by injured endothelium that might propagate the risk of thrombosis in HIT. Disruption of PF4-VWF complex formation may provide a new therapeutic approach to HIT.

Summary Background ERp57 is required for platelet function; however, whether ERp57 contributes to fibrin generation is unknown. Methods and Results Using an inhibitory anti-ERp57 antibody (mAb1), Pf4-Cre/ERp57fl/fl mice, Tie2-Cre/ERp57fl/fl mice, and mutants of ERp57, we analyzed the function of ERp57 in laser-induced thrombosis. Fibrin deposition was decreased in Pf4-Cre/ERp57fl/fl mice, consistent with a role for platelet ERp57 in fibrin generation. Fibrin deposition was further decreased with infusion of mAb1 and in Tie2-Cre/ERp57fl/fl mice, consistent with endothelial cells also contributing to fibrin deposition. Infusion of eptibifatide inhibited platelet and fibrin deposition, confirming a role for platelets in fibrin deposition. Infusion of recombinant ERp57 corrected the defect in fibrin deposition but not platelet accumulation, suggesting a direct effect of ERp57 on coagulation. mAb1 inhibited thrombin generation in vitro, consistent with a requirement for ERp57 in coagulation. Platelet accumulation was decreased to similar extents in Pf4-Cre/ERp57fl/fl mice, Tie2-Cre/ERp57fl/fl mice and normal mice infused with mAb1. Infusion of completely inactivated ERp57 or ERp57 with a non-functional second active site inhibited fibrin deposition and platelet accumulation, indicating that the isomerase activity of the second active site is required for these processes. Conclusion ERp57 regulates thrombosis via multiple targets.

Platelets are critical for haemostasis, thrombosis, and inflammatory responses, but the events that lead to mature platelet production remain incompletely understood. The bone marrow has been proposed to be a major site of platelet production, although there is indirect evidence that the lungs might also contribute to platelet biogenesis. Here, by directly imaging the lung microcirculation in mice, we show that a large number of megakaryocytes circulate through the lungs, where they dynamically release platelets. Megakaryocytes that release platelets in the lungs originate from extrapulmonary sites such as the bone marrow; we observed large megakaryocytes migrating out of the bone marrow space. The contribution of the lungs to platelet biogenesis is substantial, accounting for approximately 50% of total platelet production or 10 million platelets per hour. Furthermore, we identified populations of mature and immature megakaryocytes along with haematopoietic progenitors in the extravascular spaces of the lungs. Under conditions of thrombocytopenia and relative stem cell deficiency in the bone marrow, these progenitors can migrate out of the lungs, repopulate the bone marrow, completely reconstitute blood platelet counts, and contribute to multiple haematopoietic lineages. These results identify the lungs as a primary site of terminal platelet production and an organ with considerable haematopoietic potential.

Several CGHC motif-containing disulfide isomerases support thrombosis. We here report that endoplasmic reticulum protein 72 (ERp72), with 3 CGHC redox-active sites (ao, a, and a'), supports thrombosis. We generated a new conditional knockout mouse model and found that Tie2-Cre/ERp72fl/fl mice with blood and endothelial cells lacking ERp72 had prolonged tail bleeding times and decreased platelet accumulation in laser-induced cremaster arteriole injury and FeCl3-induced mesenteric arterial injury. Fibrin deposition was decreased in the laser injury model. Both platelet and fibrin accumulation defects were fully rescued by infusion of recombinant ERp72 containing functional a and a' CGHC motifs (ERp72(oo-ss-ss)). Infusion of ERp72 containing inactivated a and a' CGHC motifs (ERp72(ss-oo-oo)) inhibited platelet accumulation and fibrin deposition in wild-type mice. Infusion of ERp72(oo-ss-ss) into β3-null mice increased fibrin deposition in the absence of platelets. ERp72-null platelets had defective aggregation, JON/A binding, P-selectin expression, and adenosine triphosphate (ATP) secretion. The aggregation and ATP secretion defects were fully rescued by ERp72(oo-ss-ss) but partially rescued by ERp72(ss-oo-ss) and ERp72(ss-ss-oo). Aggregation and ATP secretion of human platelets was potentiated by ERp72(oo-ss-ss) but inhibited by ERp72(ss-oo-ss) and ERp72(ss-ss-oo). These data suggest that both the a and a' active sites are required for platelet function. ERp72 bound poorly to β3-null mouse platelets, and the addition of ERp72(oo-ss-ss) to human platelets generated thiols in αIIbβ3, suggesting a direct interaction of ERp72 with αIIbβ3. Defective aggregation of ERp72-null platelets was recovered by ERp72, but not other thiol isomerases. In summary, ERp72 plays a critical role in platelet function and coagulation through the a and a' CGHC motifs.

We previously described (Fuentes, J Clin Invest 2016) a chimeric protein, GPIIb/uPA-T, that provided effective thromboprophylaxis composed of an N-terminal scFv specifically to human (h) GPIIb linked to a C-terminal variant of low molecular weight urokinase containing a thrombin cleavable activating site. uPA-T prevented FeCl3-induced carotid arterial thrombosis in immunocompromized NOD/SCID, IL2 receptor γ-chain deficient (NSG) mice that had been pre-infused with human platelets (hPlts) without causing rebleeding from pre-existing tail-clips clots. However, this construct and subsequent hGPIIb-targeting scFv uPA-T chimeric constructs caused significant thrombocytopenia in hPlt-infused NSG (hPlt/NSG) mice and in hGPIIb+ mice. Therefore, we developed a new chimeric uPA-T targeting hGPIbα based on 5G6, a monoclonal antibody that binds to the hGPIbα juxtamembrane ADAM17 cleavage site and is known to not cause thrombocytopenia. The chimera 5G6/uPA-T binds to the surface of hPlts and hGPIbα-expressing mouse platelets at levels ~30% of those seen with hGPIIb/uPA-T, consistent with the difference in expression of GPIIb vs. GPIbα. 5G6/uPA-T did not reduce platelet counts in hPlt/NSG or hGPIα+ mice at doses that caused significant thrombocytopenia after infusion of GPIIb/uPA-T (1 mg/kg each, bolus + 30-minute infusion). We then tested the efficacy of 5G6/uPA-T in hPlt/NSG mice that that also carried a single mouse-to-human arginine (R) to histidine (H) substitution at position 1326 in von Willebrand factor, VWFH, created using CRISPR/Cas9 technology in NSG mice. This substitution, described by T. Diacovo (Megalion, Circulation 2011), markedly impairs binding of mouse Plts that express murine GPIbα, but increases the affinity for hPlt expressing hGPIbα. We hypothesized that such mice would allow studies of hPlt-enriched thrombi in mice. To test this hypothesis, hPlts were infused into NSG mice to achieve a circulating level of 10-20% of the total platelet pool. Following FeCl3-induced carotid arterial injury, similar numbers of hPlts were incorporated into the thrombi in wildtype VWFR/R NSG and heterozygous VWFR/H littermate NSG mice, whereas 1.4-1.8-fold more hPlts were incorporated in VWFH/H NSG littermates over the 3-minute observation period (n = 12-17, p Infusion of 5G6/uPA-T (1 mg/kg bolus+infusion) into hPlts-infused VWFH/H NSG mice prevented carotid artery thrombosis induced by FeCl3 and observed for 30 minutes as follows: In the absence of infused hPlts, 2/5 injured carotid arteries in the VWFH/H NSG mice completely occluded with a total flow volume in all vessels measured by Doppler of 1158±377 ml/min.s area under the curve (AUC). In contrast, 5/5 vessels occluded hPlts-infused VWFH/H NSG mice with an average time to occlusion of 13.0±3.4 mins and an AUC reduced to 692±75 ml/min.s (p In summary, we believe that 5G6/uPA-T combines a number of desirable features for a drug designed to prevent untoward platelet-mediated thrombosis: i. 5G6/uPA-T binds specifically to a hPlt-specific receptor, allowing targeted delivery to evolving thrombi while sparing established clots where it likely poorly penetrates into the thrombi. ii. The drug is effective even while binding to the base of the GPIbα chain near the platelet surface, where it also likely maintains GPIb/IX biology by limiting GPIbα cleavage, and does not cause thrombocytopenia. iii. Finally, 5G6/uPA-T delivers a form of urokinase that needs the co-presence of thrombin to become activated further limiting its activity to sites of evolving thrombi. These properties were demonstrated in an in vivo model that selectively incorporates hPlts. We believe that 5G6 may have clinical application in settings where there is a high risk for thrombosis, such as maintaining stent patency, while avoiding untoward lysis of previously established clots. Disclosures Li: Neoletix: Consultancy, Equity Ownership. Poncz:Incyte Corporation: Consultancy, Research Funding.

Understanding platelet biology has been aided by studies of mice with mutations in key megakaryocytic transcription factors. We have shown that point mutations in the GATA1 cofactor FOG1 that disrupt binding to the nucleosome remodeling and deacetylase (NuRD) complex have erythroid and megakaryocyte lineages defects. Mice that are homozygous for a FOG1 point mutation (ki/ki), which ablates FOG1-NuRD interactions, have platelets that display a gray platelet syndrome (GPS)–like macrothrombocytopenia. These platelets have few α-granules and an increased number of lysosomal-like vacuoles on electron microscopy, reminiscent of the platelet in patients with GATA1-related X-linked GPS. Here we further characterized the platelet defect in ki/ki mice. We found markedly deficient levels of P-selectin protein limited to megakaryocytes and platelets. Other α-granule proteins were expressed at normal levels and were appropriately localized to α-granule–like structures. Treatment of ki/ki platelets with thrombin failed to stimulate Akt phosphorylation, resulting in poor granule secretion and platelet aggregation. These studies show that disruption of the GATA1/FOG1/NuRD transcriptional system results in a complex, pleiotropic platelet defect beyond GPS-like macrothrombocytopenia and suggest that this transcriptional complex regulates not only megakaryopoiesis but also α-granule generation and signaling pathways required for granule secretion.

Contraction of blood clots is necessary for hemostasis and wound healing and to restore flow past obstructive thrombi, but little is known about the structure of contracted clots or the role of erythrocytes in contraction. We found that contracted blood clots develop a remarkable structure, with a meshwork of fibrin and platelet aggregates on the exterior of the clot and a close-packed, tessellated array of compressed polyhedral erythrocytes within. The same results were obtained after initiation of clotting with various activators and also with clots from reconstituted human blood and mouse blood. Such close-packed arrays of polyhedral erythrocytes, or polyhedrocytes, were also observed in human arterial thrombi taken from patients. The mechanical nature of this shape change was confirmed by polyhedrocyte formation from the forces of centrifugation of blood without clotting. Platelets (with their cytoskeletal motility proteins) and fibrin(ogen) (as the substrate bridging platelets for contraction) are required to generate the forces necessary to segregate platelets/fibrin from erythrocytes and to compress erythrocytes into a tightly packed array. These results demonstrate how contracted clots form an impermeable barrier important for hemostasis and wound healing and help explain how fibrinolysis is greatly retarded as clots contract.

The release of biologically intact platelets from in vitro-grown megakaryocytes remains one of the challenges in an attempt to replace donor-derived platelets with platelets prepared from in vitro-grown megakaryocytes. Thrombopoiesis, the process by which megakaryocytes release platelets, is thought to occur as megakaryocytes exit from the medullar space and transit into the blood stream. Two-photon fluorescence microscopy studies by several groups have documented megakaryocytes transitioning into the blood stream and extending mostly a single protrusion that appears to release large cytoplasmic fragments downstream. These studies have been widely interpreted to support platelet release in the bone marrow environment. A counter proposal made by Dr. William Howell over 75 years ago is that either whole megakaryocytes or large cytoplasmic fragments circulate to the lungs and release platelets in the lungs. Studies by our group infusing in vitro-grown megakaryocytes (both human and mouse) have shown that infused megakaryocytes do indeed become entrapped in the lungs and release platelets. These in vivo-released platelets have a much longer half-life than in vitro-prepared platelets from static cultures and have the same size distribution and nearly the same functionality as infused donor-derived platelets. Using confocal intravital microscopy, we now directly visualized the lungs of recipient NOD-SCID interferon 2 receptor γ-deficient mice during the infusion of calcein-loaded CD34+-derived megakaryocytes. Infused human megakaryocytes were immediately arrested in the pulmonary bed in vessels ~50 µm in diameter. Each cell then extended several distinct protrusions winding down presumed pulmonary capillaries that are presumably wrapped around alveoli. Some of these protrusions reached 200-300 µm in length and assumed the appearance of beads on a string. Consistent with our recently published data, this thrombopoiesis process appears to take 30 minutes to be near-complete, similar to the timeframe we showed for detecting newly released human platelet-like particles after megakaryocyte infusion. At 30 minutes, remaining bodies of the megakaryocytes are still present and we presume these are mostly the remaining nuclei. To better define whether the pulmonary bed is unique for releasing platelets, we also infused megakaryocytes intra-arteriole rather than intravenously to have the megakaryocytes encounter other organ capillary beds before the lung. These studies showed poor platelet release compared to parallel studies in mice receiving the megakaryocytes intravenously. Many of the intra-arterial infused megakaryocytes were entrapped in the spleen and very few were notable in other organs, including the lungs, liver and kidney. Our studies showed that megakaryocytes can shed platelets in the lungs where they may take advantage of the unique three-dimensional organization of the pulmonary vascular bed, flow conditions, vascular surface receptors and glycocalyx as well as a sudden shift from hypoxic to normoxic conditions. Whether these features of the pulmonary vascular bed can be simulated ex vivo and whether this will enhance true platelet release from megakaryocytes in vitro needs to be examined. Disclosures No relevant conflicts of interest to declare.

The integrin family is composed of a series of 24 αβ heterodimer transmembrane adhesion receptors that mediate cell-cell and cell-extracellular matrix interactions. Adaptor molecules bearing immunoreceptor tyrosine-based activation motifs (ITAMs) have recently been shown to cooperate with specific integrins to increase the efficiency of transmitting ligand-binding–induced signals into cells. In human platelets, Fc receptor γ-chain IIa (FcγRIIa) has been identified as an ITAM-bearing transmembrane receptor responsible for mediating “outside-in” signaling through αIIbβ3, the major adhesion receptor on the platelet surface. To explore the importance of FcγRIIa in thrombosis and hemostasis, we subjected FcγRIIa-negative and FcγRIIa-positive murine platelets to a number of well-accepted models of platelet function. Compared with their FcγRIIa-negative counterparts, FcγRIIa-positive platelets exhibited increased tyrosine phosphorylation of Syk and phospholipase Cγ2 and increased spreading upon interaction with immobilized fibrinogen, retracted a fibrin clot faster, and showed markedly enhanced thrombus formation when perfused over a collagen-coated flow chamber under conditions of arterial and venous shear. They also displayed increased thrombus formation and fibrin deposition in in vivo models of vascular injury. Taken together, these data establish FcγRIIa as a physiologically important functional conduit for αIIbβ3-mediated outside-in signaling, and suggest that modulating the activity of this novel integrin/ITAM pair might be effective in controlling thrombosis.

Heparin induced thrombocytopenia (HIT) is an iatrogenic antibody-mediated disorder with a paradoxically high propensity for thrombosis. We have shown previously that human HIT IgGs and the HIT-like monoclonal antibody (MAb) KKO bind to platelet factor 4 (PF4) complexed with glycosaminoglycans (GAGs) on the surface of platelets and monocytes, initiating cell activation in vitro, thrombocytopenia in a transgenic mouse model, and thrombus formation in a laser microvascular injury model in vivo even in the absence of exogenous heparin. Monocytes bind PF4 and HIT Ab more readily than platelets because they express higher affinity GAGs, heparan sulfate and dermatan sulfate, in addition to chondroitin sulfate found on both cell types. To study changes in the structure of the monocytes that accompany HIT, we used scanning electron microscopy, confocal microscopy and flow cytometry to characterize the morphology and function of isolated human monocytes and mouse transgenic Fcg receptor IIA positive (FcγRIIA+) or wt (FcγRIIA-) monocytes in the absence or presence of platelets. We show by scanning electron microscopy that upon binding of pathogenic HIT Abs to PF4/GAG complexes on FcgRIIA expressing monocytes, they initiate profound remodeling of the cell membrane. Addition of 100 μg/ml recombinant human PF4 in the absence of HIT Abs initiates the activation process with the appearance of 177 ± 53 nm "knobs" on the surface of 70% of monocytes. Subsequent addition of the HIT-like monoclonal antibody KKO at 50 μg/ml dramatically alters the cellular surface with the appearance of large 701 ± 208 nm membrane "blebs" that were not seen on FcγRIIA-mouse monocytes. These large, membrane-associated structures likely engage FcγRIIA, clustering them in proximity to cell-bound immune complexes, which promotes cell activation that leads to thrombosis. These blebs increase in size over time and are then shed from the cells as monocyte-derived microparticles, which self-aggregate. As a result of shedding of these blebs, the monocytes lose much of their typical ruffled surface (only 67% of monocytes maintain ruffles in the presence of PF4 plus KKO, compared to 97% of control monocytes) and appear smoother, sometimes with pores indicating degranulation. In the presence of platelets, monocytes exposed to PF4 and KKO formed heterocellular aggregates in addition to these subcellular changes. In contrast to KKO, addition of the non-pathogenic MAb RTO not only did not induce blebbing, but largely inhibited PF4-induced changes in the monocyte surface. This suggests that RTO might prevent monocyte activation by interfering with PF4 tetramerization. Structural analysis of the shed microparticles by microscopy revealed that they had an average diameter of 356 ± 307 nm, with many larger particles and aggregates. Flow cytometry confirmed that the shed particles contain cell membrane lipids and receptors. Confocal microscopy showed uniform binding of labeled PF4 to the monocyte cell membrane followed by rapid clustering into large complexes after the addition of KKO, but not RTO. These studies affirm the centrality of cell surface PF4/GAG complexes in the pathogenesis of HIT and provide quantitative morphometric characteristics of the changes in the monocyte membrane structure. We propose that PF4 released from activated platelets binds to the surface of GAG-expressing monocytes in vivo, forming clusters of PF4/GAG complexes that likely promote antibody binding and cause monocyte activation through FcγRIIA along with large-scale remodeling of the cell membrane and shedding of procoagulant microparticles. Disclosures No relevant conflicts of interest to declare.

Replicating the complexities of the human blood vessel include the establishment of a 3-D confluency of viable endothelial cells (ECs) on an appropriate matrix, use of human whole blood or specific components of blood, varied shear stresses, and the induction of a localized and controlled injury within the observable field to understand and intervene in hemostatic events. This array of complexities have made vascular modeling an important unmet challenge. Such a model would enhance our understanding of the pathogenesis of diverse coagulation disorders, such as the prothrombotic disorder HIT. In HIT, the platelet-rich clots lead to its other designation as the "white clot syndrome". To provide an improved injury model, we adapted a hematoporphyrin-photochemical injury using a Fluxion Bioflux microfluidic system. When illuminated with 405 nm light, hematoporphyrin releases reactive oxygen species, inducing localized EC injury within the exposed field, but without denuding ECs. Unlike rose bengal, hematoporphyrin does not cause fluorescent interference with quantitative analysis of the developing thrombus. We propose that the model permits refined analysis of ECs transitioning from areas of quiescence to injury to quiescence, allowing us to localize and quantify the contribution of various components to the growing thrombus. In HIT, patients form antibodies to complexes of the platelet-specific chemokine, platelet factor 4 (PF4), and negatively-charged molecules such as infused heparin and heparans found on the surface of platelets and monocytes. ECs also bind PF4 immune complexes due to their highly negatively charged glycocalyx-rich surface. Prior murine cremaster laser injury studies showed that HIT antibodies bind predominantly to the EC surface rather than platelet within the thrombus itself. Was this observation related to the nature of the cremaster injury? Would antibody binding to the EC lining also be important in a wholly human HIT detection system? Using the described photochemical microfluidic system, we created a localized injury in human umbilical vein EC-lined channels through which we flowed whole human blood. Activated platelets established growing aggregates at the site of EC injury, releasing more PF4 that then bound to non-activated ECs downstream of the injury. Whole blood containing a HIT-like antibody to simulate the prothrombotic state of HIT was then flowed over this injured vasculature. HIT antibodies and then platelets bound sequentially to this new site of HIT antigen, allowing the thrombus to propagate downstream. We have named this phenomenon "rolling barrage" and suggest that a key part of the prothrombotic nature of HIT lies in antibody-mediated activation of downstream EC with subsequent thrombus propagation. HIT often occurs in the setting in surgery, which might prime EC dysfunction. We therefore treated the ECs (TNF-α) prior to injury and introducing the HIT-like antibody. TNF-α activated the EC lining, leading to loss of the anticoagulant EC surface, which enhanced clot formation downstream of the site of photochemical injury. Therefore, in a prothrombotic state such as HIT, we propose that a local injury acts as a nidus for thrombus initiation. The procoagulant process spreads distally in part because of released PF4 adhering to the downstream EC glycocalyx, which is exacerbated by mediators of inflammation. We anticipate that the described model can be used to study novel interventions to block this cycle in a wholly human system with control over the contribution of individual cellular elements, and will further understanding of the importance of this mechanism in other prothrombotic disorders. Disclosures No relevant conflicts of interest to declare.

Protein disulfide isomerase (PDI) catalyzes thiol-disulfide exchange reactions that are critical for protein function. PDI has two distinct CGHC redox-active sites, but nothing is known about which site is important in physiologic reactions including thrombosis. To study the role of PDI and its active sites in thrombosis we used two genetically-modified models, Mx1-Cre/PDIfl/fl mice with blood cells and vessel wall cells lacking PDI, and transgenic PDI(ss-oo) mice with PDI lacking the second CGHC active site. Using laser-induced cremaster arteriole injury fibrin deposition was decreased in both mouse models. Both mouse models showed attenuation of platelet accumulation using laser arterial injury and FeCl3-induced mesenteric artery injury. The defects in fibrin deposition and platelet accumulation were rescued by infusion of recombinant PDI(oo-ss) containing only a functional second active site, confirming the second active site of PDI was critical for both fibrin formation and platelet accumulation. In vitro studies showed that platelet aggregation depended on the second CGHC motif of PDI, and that binding of PDI protein to platelets depended on the presence of the αIIbβ3 integrin. Studies on platelet secretion revealed that P-selectin expression and ATP release were dependent on a functional second active site of PDI. Treatment of the platelets with eptifibatide did not affect P-selectin expression. Together these findings suggest the second active of PDI catalyzes a reaction in αIIbβ3 supporting platelet aggregation and a reaction in a non-αIIbβ3 substrate supporting secretion. In vivo studies on coagulation showed that PDI infusion rescued fibrin formation in eptifibatide-treated mice in which platelet accumulation was completely blocked, suggesting that PDI directly contributes to activation of coagulation. In summary, our results indicate that the second CGHC active site of PDI has a role in platelet function and coagulation. Targeting this site may have a dual inhibitory effect on thrombosis by decreasing both platelet and fibrin accumulation. Disclosures No relevant conflicts of interest to declare.

Heparin-induced thrombocytopenia (HIT) is a life- and limb-threatening thrombotic disorder that develops after exposure to heparin, often in the setting of inflammation. We have shown previously that HIT is associated with antibodies to complexes that form between platelet factor 4 and glycosaminoglycan (GAG) side chains on the surface of platelets. However, thrombosis can occur in the absence of thrombocytopenia. We now show that platelet factor 4 binds to monocytes and forms antigenic complexes with their surface GAG side chains more efficiently than on platelets likely due to differences in GAG composition. Binding to monocytes is enhanced when the cells are activated by endotoxin. Monocyte accumulation within developing arteriolar thrombi was visualized by situ microscopy. Monocyte depletion or inactivation in vivo attenuates thrombus formation induced by photochemical injury of the carotid artery in a modified murine model of HIT while paradoxically exacerbating thrombocytopenia. These studies demonstrate a previously unappreciated role for monocytes in the pathogenesis of arterial thrombosis in HIT and suggest that therapies targeting these cells might provide an alternative approach to help limit thrombosis in this and possibly other thrombotic disorders that occur in the setting of inflammation.

Ex vivo-generated (EV) platelets beginning with embryonic stem cells or induced pluripotent stem cells (iPSCs) or hematopoietic progenitors cells (HPCs) may have clinical utility over donor-derived platelets, and efforts to produce such EV-platelets have been pursued in several laboratories under static megakaryocyte (Meg) culture conditions. Success in generating these has been reported, even demonstrating EV-platelet incorporation into growing thrombi in murine models. We have pursued an alternative strategy for thrombopoiesis using EV-Megs, grown from either human adult HPCs or from iPSCs or fetal livers, and directly infusing them into NOD-SCID gamma-interferon-deficient (NSG) mice. These studies were based on our prior observation that infused murine EV-Megs into wildtype mice are entrapped in the pulmonary bed and over the subsequent 1-4 hours release a wave of functional platelets at a significant level. We now show that infusion of human EV-Megs do the same in NSG mice, but resulting in two different pools of derived platelets: (1) A pool of young (as determined by thiazole orange staining) platelets having the same bell-shaped size distribution was seen as after infusion of human donor-derived platelets in these mice. These platelets take several hours to appear, but then have the same half-life as donor-derived platelets. These platelets are derived from the infused EV-Megs and were termed in vivo-generated (IV)-platelets. (2) A second pool of mostly older platelets was present that originated during the static growth of the EV-Megs, and these EV-platelets varied widely in size and age. Initially, these platelets accounted for a third of all the human platelets seen. Unlike IV-platelets, EV-platelets are immediately present and circulate with a markedly short half-life of 2-3 hours unless the recipient NSG mice were pre-treated with clodronate-ladened liposomes to delete their macrophage pools. Rapid removal of EV-platelets by macrophages is due to their being preactivated as determined by surface P-selectin expression in whole mice blood. These EV-platelets also had very limited further responsiveness to convulxin activation. On the other hand, human IV-platelets were quiescent prior to agonist stimulation in whole mice blood and responded strongly to agonist, similar to human donor-derived platelets infused into NSG mice. The IV-platelets were also selectively incorporated into cremaster arteriole laser injury thrombi over EV-platelets. Finally, directly harvested “platelets” from EV static-grown Megs were isolated and analyzed both in vitro and in vivo. Only a third of these particles are CD41+/CD42+ platelets and approximately half are actually CD41-/CD42-. Both pools showed the same wide size distribution in vitro and in vivo after infusion into mice. The CD41+/CD42+ fraction behaved just as the EV-platelets, but the CD41-/CD42- fraction half-life was unaffected by pre-treatment with clodronate-ladened liposomes. In summary, infused human Megs grown under static growth conditions released platelets in a recipient mouse’s lung with features characteristic of donor-derived platelets. On the other hand, “platelets” harvested from the same Megs were predominantly not even platelets as measured using CD41/CD42 markers. The portion that were CD41+/CD42+ platelets were preactivated, poorly responsive to agonists, and cleared rapidly. These findings set a standard on how to judge the potential clinical value of platelets derived from EV-Megs and also raise concerns whether direct visual imaging of “platelet release” in static culture is biologically meaningful given that most particles released were not CD41+/CD42+ platelets, and the ones that were CD41+/CD42+ were mis-sized and functionally limited. Disclosures No relevant conflicts of interest to declare.

The most feared feature of HIT is antibody-mediated thrombosis. We have shown that this prothrombotic state is related to binding of platelet factor 4 (PF4), a chemokine densely packaged into platelet alpha-granules, to surface glycosaminoglycans (GAGs) expressed on hematopoietic and vascular cells. PF4/GAG surface complexes are recognized by HIT antibodies, activating the targeted cells. Unlike platelets that express only low-affinity chondroitin sulfate surface GAGs, endothelial cells (EC) express a glycocalyx enriched in heparan sulfate, which has higher affinity for PF4, potentially increasing their propensity to become a target for immune injury leading to thrombosis. We examined the details of the development of in situ thrombi using the cremaster arteriole laser injury model beginning with transgenic mice expressing only human PF4 (hPF4+), but lacking FcγRIIA. These mice do not develop thrombocytopenia or thrombosis when injected with the HIT-like monoclonal antibody KKO or IgGs isolated from patients with HIT. In these mice, antigenic PF4/GAG complexes were recognized by KKO at sites of vascular injury even in the absence of infused heparin. In fact, infusion of sufficient heparin dissociated PF4 from sites of injury, consistent with its higher affinity for PF4 than cell surface GAGs. This suggests that antigenic PF4/GAG complexes normally develop intravascularly whenever thrombus formation occurs, yet these complexes do not typically initiate antibody-mediated thrombosis. Real-time confocal imaging of injured vessels revealed that PF4 first bound almost exclusively to the peri-injury endothelium. This was especially evident immediately upstream of the thrombus where turbulent blood flow may lead to platelet degranulation and subsequent adherence of the released PF4 to the glycocalyx. Beginning approximately two minutes post-injury, binding of KKO, presumably to PF4/GAG complexes on platelets, is seen at the interface between the shell and core of the thrombus. We then repeated these same studies in hPF4+/FcγRIIA+ mice, where infused KKO or HIT IgGs leads to significant thrombocytopenia and widespread development of thrombi as in HIT. Similar adherence of PF4 to the peri-injury EC and then to the core/shell interface of the thrombus as seen in the hPF4+ mice, but the changes were more extensive in hPF4+/FcγRIIA+ mice after KKO infusion and often lead to vascular occlusion. To further define the basis of the prothrombotic state in HIT and to extend our studies to a human system, we examined thrombus formation in HIT in a novel microfluidic system in which vascular injury was induced in an upstream portion of a human umbilical vein EC-lined channel by reactive oxygen species generated through excitation of infused hematoporphyrin by blue light (490 nm). Following infusion of human blood, platelets accumulated and released PF4, which bound the injured endothelium, while the downstream endothelium remained quiescent. Addition of KKO to the infused whole blood lead to a HIT-like state with marked increase in platelet adhesion and binding of PF4 to the injured endothelium, but binding of PF4 now spread downstream of the boundary between injured and uninjured endothelium. This was followed by downstream platelet adhesion and often occlusion of the channel. We proposed that this spread in EC injury was a result of a “rolling barrage” of PF4 released from platelets binding to the injured patch of EC complexing to the downstream glycocalyx on the non-injured endothelium followed by KKO binding and subsequent endothelial activation. The newly activated ECs bound additional platelets and the process repeats, rolling downstream and extending thrombus growth. Thus, these studies provide important new insights into the local sequence of events that propagate clots in HIT: Targeting of the endothelial glycocalyx by HIT antibodies is a major contributor to the prothrombotic state. Platelets adherent to the site of original injury release PF4, which then binds to downstream EC glycocalyx and initiates repetitive cycles of PF4 binding, EC activation and platelet adherence, and further release of PF4 that propagates growth of thrombi to previously uninvolved vasculature. Disclosures No relevant conflicts of interest to declare.

Two major clinical limitations to the use of thrombolytic agents are their short half-life and lack of targeting specificity. We aimed to circumvent these limitations by targeting a platelet-bound, thrombin-activatable, low molecular weight urokinase, termed PLT/uPA-T, to nascent thrombi by a combination of two strategies: (1) Attaching the drug through its N-terminal scFv portion to human αIIb/β3 on the surface of platelets with nM affinity. (2) Protecting the uPA from rapid activation/inactivation in the circulation while simultaneously requiring its activation by thrombin through a two amino acid deletion at the plasmin cleavage site that concurrently creates a thrombin cleavage/activation site. These two properties constrain the activity of PLT/uPA-T on mature clots, which express low levels of thrombin and transiently recruit only a few new platelets to the shell rather than the core of the thrombus. These two properties of PLT/uPA-T also enhance the drug’s lifespan by attaching it to the platelet cell surface and preventing its inactivation before reaching its intended target. PLT/uPA-T binds specifically to human platelets and to hαIIb mouse platelets that transgenically expressed human (h) αIIb on its surface. In hαIIb mice, the half-life of retro-orbitally infused PLT/uPA-T was ~2 hours, ~100-fold longer than similarly infused uPA-T and did not cause any spontaneous bleeding or lower platelet counts 4 to 24 hours later. We now report two in vivo models to test the efficacy of the PLT/uPA-T as a thromboprophylactic agent versus uPA-T, taking into account the much shorter circulating half-life of the latter. In the hαIIb mice model, a tail-vein clipping model was done to represent the “mature clot” as follows: following clipping, blood was collected into 37°C water for 10 minutes. The tail was then removed from the water and a bolus of PLT/uPA-T (0.5 µg/g mouse) injected retro-orbitally followed by a continuous infusion of the same dose over the next 30 minutes. uPA-T was similarly infused but both bolus and infusion were given at 10-fold higher doses. A no-drug treatment control was also included. After the drug infusion was started, the tail was placed into fresh 37°C water and bleeding was documented from these “mature clots” over the ensuing 30 minutes. To study “nascent thrombi”, mice were bolused/infused with same drug regimens, and a FeCl3 carotid artery injury study was performed contemporaneously. PLT/uPA-T was as effective at 1/10th the dose as uPA-T at preventing these “nascent thrombi” (FeCl3 injuries), but did not cause bleeding from “mature clots” (tail clippings) relative to the no-treatment control, while the uPA-T treatment lead to ~5-fold greater rebleeding compared to the no-treatment control (p 10 animals per arm. The second in vivo model targeted human platelets infused into immunocompromized NOD-SCID γ-interferon-deficient (NSG) male mice to generate a calculated 10% of all the circulating platelets being human at the initiation of the studies. The pre-drug “mature clots” and the post-drug “nascent thrombi” were both arteriolar laser cremaster injuries. We enumerated mouse platelets incorporated into these thrombi over time. There was an ~50% decrease in the size of laser-induced post-drug “nascent thrombi” after PLT/uPA-T or a 10-fold higher dose of uPA-T relative to no drug treatment. However, PLT/uPA-T did not affect the size of the laser-induced pre-drug “mature clots” relative to no drug-treatment, while there was a decrease in size of the mature clots after treatment with uPA-T. These studies describe two preclinical models of comparative thromboprophylactic efficacy and safety that are independent of drug half-life. Our studies demonstrate that a combination of platelet-targeting and need for thrombin-activation makes PLT/uPA-T a very potent and targeted thrombolytic agent to prevent new thrombus formation, while leaving older, hemostatic clots intact. Disclosures No relevant conflicts of interest to declare.

Background Contraction of blood clots is necessary for hemostasis, wound healing and to restore flow past obstructive thrombi. However, little has been known about the structure of contracted clots and mechanisms of contraction. Erythrocytes, biconcave cells that are highly deformable to allow their passage through the microvasculature, are abundant in venous thrombi, and to a lesser extent in arterial thrombi. Erythrocytes promote hemostasis, but their participation in clot contraction has not been reported. Here we study the mechanisms of clot contraction and the roles of erythrocytes, platelets and fibrin, and show that erythrocyte shape change into compressed polyhedrocytes allows tight packing consistent with the major function of clots to stem bleeding. Methods Whole blood was clotted by recalcification and addition of thrombin or kaolin, while following the process of clotting, including contraction, with a new technique using T2 magnetic resonance. We examined the structure and composition of contracted whole blood clots by scanning electron microscopy and confocal light microscopy. Results Contracted clots display a remarkable structure, with a close-packed, tessellated array (or mosaic tiling of space) of compressed polyhedral erythrocytes (called polyhedrocytes) on the interior and a meshwork of fibrin and platelet aggregates on the exterior. Little fibin and few platelets were found on the interior of the contracted clots. The same results were obtained with both thrombin and kaolin as activators of clotting and also with reconstituted human blood and clots prepared from mouse blood. Confocal microscopy of hydrated clots confirms the results of scanning electron microscopy. The mechanical nature of this shape change was confirmed by polyhedrocyte formation from the forces of centrifugation of blood without clotting. Platelets (with their cytoskeletal motility proteins) and fibrin(ogen) (as the substrate bridging platelets for contraction) are required to generate the forces necessary to segregate platelets/fibrin from erythrocytes and to compress erythrocytes into a closely packed polyhedral array. To assess the density of packing of the polyhedral erythrocytes, we replaced the water surrounding the clots with D2O and observed by T2 magnetic resonance that hydrogen/deuterium exchange for the contracted clots was very slow, consistent with their very tightly packed, almost impermeable structure. The same polyhedrocyte structures were observed from in vivo thrombi aspirated by cardiologists from the coronary arteries of ST-elevation myocardial infarction patients. Summary/Conclusions We have observed a previously undiscovered, naturally occurring erythrocyte function and morphology, closely packed polyhedra, in contracted clots and thrombi, and an unexpected spatial redistribution of platelets and fibrin that occurs during contraction. Clot contraction is an essential part of hemostasis, since both human genetic disorders of platelet myosin IIA and megakaryocyte myosin IIA-knock out mice show a bleeding phenotype. These observations on contracted clots imply that they are stiff, rigid structures that can form an impermeable, watertight seal. On the one hand, contraction of clots within the vasculature may relieve obstruction of blood vessels and allow recanalization, especially in the venous system. On the other hand, these results account for long-standing clinical observations that fibrinolysis is greatly prolonged following clot contraction, since perfusion or diffusion of lytic enzymes into these tightly packed polyhedral erythrocytes would be nearly impossible. These results suggest a vital role for erythrocytes and clot contraction in hemostasis and wound healing. Disclosures: No relevant conflicts of interest to declare.

Thrombopoiesis is the process by which megakaryocytes (Megs) release platelets (Plts), but issues remain as to the detailed in vivo mechanisms underlying this process. We now report new insights into this process by studying infused human Megs into immunocompromized NOD/SCID, gamma-interferon deleted (NSG) mice. Prior in situ microscopy has suggested that Megs release varied-size cytoplasmic fragments up to whole Megs in size into the medullary vascular space. Other studies have suggested that at least a portion of thrombopoiesis occurs by Megs lodged in the lungs. We previously infused ex vivo-generated murine Megs into mice and found that these Megs become entrapped in the animals’ lungs, and in Disclosures: No relevant conflicts of interest to declare.

HIT is an antibody-mediated disorder characterized by thrombocytopenia and thrombosis. Activation of platelet via FcγRIIA by HIT antibodies complexed to surface-bound platelet factor 4 (PF4) contributes to the prothrombotic state. However, using an in vitro microfluidic whole human blood model of HIT, we have found through depletion and repletion studies that activation of monocytes through Syk-kinase is a key step in generating a prothrombotic milieu. Here we define the Fcγ receptor(s) upstream of Syk that mediate the thrombotic signal in monocytes. Monocytes isolated from normal healthy volunteers were incubated with 50 µg/ml of blocking antibodies against FcγRIIA (CD32, clone IV.3) and/or against FcγRI (CD64, clone 10.1) or against FcγRIII (CD16, clone 3G8), washed to remove unbound antibody and added to monocyte depleted-whole blood. These monocyte-repleted samples were then stimulated with PF4 (10 µg/ml) and the HIT like monoclonal antibody KKO (50 µg/ml) for 30 min. After recalcification (5 mmol CaCl2), samples were perfused over a von Willebrand factor-coated BioFlux microfluidic channel at a shear stress of 20 dyne/cm2 at 37°C for 15 min. Platelet adhesion was quantified by fluorescence microscopy after adding Calcein-AM (3 µM), and fibrin was visualized by adding Alexa 561-labeled fibrinogen (1.5 μg/ml) to the whole blood prior to the perfusion. Selective inhibition of FcγRIIA on monocytes led to an ∼40% inhibition of fibrin deposition and comparable reduction in the formation of “coated” platelets relative to samples repleted with non-inhibited monocytes. Flow cytometry studies showed that these observations were not due to leaching of IV.3 antibody off the monocytes with subsequent blocking of platelet FcγRIIA. In contrast, selective blockade of FcγRI or FcγRIII in depletion/repletion studies or in whole blood did not significantly reduce platelet adhesion, formation of coated platelets, or fibrin accumulation. However, blocking FcγRI did decrease tissue factor (TF) activity on monocytes stimulated with PF4 and KKO and this effect was additive to FcγRIIA inhibition. In summary, our data show that monocyte-bound HIT IgG antibodies contribute to the prothrombotic state by activating FcγRIIA directly and indirectly by generating TF though FcγRI. We speculate that this activation of monocytes contributes to platelet activation and monocyte microparticle formation demonstrated previously. Drugs that target FcγRI and FcγRIIA on monocytes may help prevent thrombosis in HIT without causing platelet dysfunction or increasing the risk of bleeding. Disclosures: No relevant conflicts of interest to declare.

Abstract 3450 Our goal is to generate sufficient PLTs from ex vivo-generated MKs for clinical utility in PLT transfusions. A critical step in this process begins with ex vivo-generated hMKs and deriving clinically relevant PLTs. We reported that infused mature, high-ploidy murine (m) MKs derived from fetal liver (FL) cells increased PLT counts in recipient mice in a clinically relevant fashion, thus avoiding the need to generate ex vivo functional PLTs. We examined whether this strategy applies to hMKs derived from FL cells (gestational age, 17–21 weeks) and bone marrow (BM) cells in a xeno-transfusion model using immunodeficient NOD/SCID/IL-2Rγcnull (NSG) mice. Infused hPLTs isolated from blood had a half-life (T1/2) of 10 hours (hrs), compared to 24 hrs for infused murine PLTs. The hPLTs were functional in NSG mice as demonstrated by their incorporation into growing thrombi in situ. Both hFL hematopoietic mononuclear cells and hBM-CD34+ cells were cultured in serum-free media supplemented with optimized cytokine cocktails to generate hMKs. In contrast to the murine studies where the FL cell-derived mMKs were the most efficient source of derived mPLTs, FL cell-derived hMKs had low ploidy (0% ≥ 8N ploidy), gave rise to ∼16 PLTs/infused hMK, and had a short T1/2 (6 hrs). In contrast, 17% of hBM cell-derived MKs had a ploidy of ≥ 8N, and after infusion into NSG mice, resulted in a wave of MKs transiently entrapped in the pulmonary microvasculature and then over ∼0.5–3 hrs released PLTs with a T1/2 of 10 hrs, comparable to infused hPLTs. Maximally, we achieved a level of 5% of circulating total PLTs being derived from human cells with ∼32 PLTs/infused hMK. These hPLTs were normal in size, displayed normal levels of surface markers, were functional, and incorporated into growing thrombi. One strategy to increase hPLT yield is to expose developing MKs to drugs reported to increase MK maturation, thrombopoiesis, and/or facilitate hematopoietic progenitor cell expansion. Such drugs include dimethylfasudil (diMF) (an inhibitor of several kinases involved in polyploidization), UNC0638 (a G9a histone methyltransferase inhibitor), SR1 (an AhR antagonist), and nicotinamide (a sirtuin histone/protein deacetylases inhibitor). Although diMF promoted size and polyploidization of hMKs, diMF markedly worsened yield of PLTs/infused hMK and decreased PLTs T1/2 in vivo. UNC0638 led to significant cell expansion, but lowered hMKs ploidy and PLTs/infused hMK yield. Nicotinamide increased maturation, size and polyploidization of hMKs, but PLT release following MK infusion needs further study. Of note, SR1 that has been reported to promote the expansion of human HSC, not only increased size and ploidy of hMKs, but also hPLT release in vitro and in vivo. SR1-treated hMKs resulted in a 3-fold increased yield of normal size, T1/2 and functional PLTs/infused hMK compared to a DMSO-treated control. In summary, like mMKs, infused hMKs into mice release PLTs in the pulmonary vasculature though at a lower efficiency. Released hPLTs were functional and T1/2 was as expected. diMF enhanced MK ploidy, but worsened PLT yield and T1/2, while an AhR antagonist SR1 that also improved MK ploidy appears to markedly enhance yield of PLT/infused hMK, while maintaining T1/2. The ability of SR1 to enhance PLT release from induced pluripotent stem cells (iPSCs)-derived MKs remains to be tested, but this drug appears to be a strong candidate for a therapeutic strategy to take ex vivo-grown hMKs and generate PLTs in clinical relevant numbers. Disclosures: No relevant conflicts of interest to declare.

Abstract 1171 Thrombolytic therapies are limited in their use to acute life- or limb-threatening thrombosis due to the high risk of significant bleeding. To overcome this limitation, we propose a novel strategy incorporating two targeting features: 1) an N-terminal single-chain antibody variable region (scFv) chain that binds with high affinity to human platelet alphaIIb, and 2) a C-terminal thrombin-specific activatable low molecular weight urokinase (uPA-T) that has its plasmin-activation site replaced by a thrombin cleavage site. We anticipate that such pro-drugs would preferentially bind and become activated at sites of active clot propagation. Two distinct anti-alphaIIb scFvs were studied: one (312.8) derived from a monoclonal antibody that bound human activated or quiescent alphaIIb whether complexed to human or mouse beta3, and one (LIBS) that bound preferentially to activated human or mouse alphaIIb bound to either human or mouse beta3. We anticipated that the 312.8/uPA-T chimera would have a more prolonged half-life, but that the LIBS version would be more effective at the nascent clot where activated platelets predominate. All proteins studied (uPA-T, 312.8/uPA-T and LIBS/uPA-T) were expressed and isolated from Drosophila S2 insect cells. Flow cytometric studies confirmed the expected binding of the chimera proteins to resting and activated human platelets, wildtype (WT) mouse platelets and mouse platelets that expressed only human alphaIIb/mouse beta3 (haIIb/mb3) on their surface. Neither chimeric protein interfered with platelet aggregation stimulated by adenosine diphosphate (10 μM). Further studies using diverse serine proteases confirmed that induction of fibrinolytic activities of both chimeras was thrombin-specific, and both were activated by thrombin to the same extent as isolated uPA-T. In haIIb/mb3 mice, both flow cytometric studies and tracking of 125I-labeled chimeric proteins showed 312.8/uPA-T had a longer half-life than uPA-T or LIBS/uPA-T. Ex vivo studies of thromboprophylactic efficacy of the chimeric proteins relative to uPA-T were performed using a microfluidic system with the surfaces coated with species-specific von Willebrand Factor. Platelets were isolated from whole human or haIIb/mb3 murine blood, exposed to a fibrinolytic agent or control, gel filtered to remove unbound drug, and added back to reconstitute whole blood. 312.8/uPA-T decreased platelet aggregation and virtually eliminated fibrin formation at drug concentrations Disclosures: Cines: Amgen: Consultancy; Genzyme: Consultancy; Sanofi: Consultancy; Eisai: Consultancy.

Abstract 270 HIT is an immune thrombocytopenic disorder associated with a high risk of thrombosis. Mechanistic studies have focused on circulating platelet factor 4 (PF4)/heparin complexes and on the subsequent activation of platelets. We and others have shown that binding of PF4 to cell surface glycosaminoglycans (GAGs) not only makes platelets critical targets in the pathogenesis of HIT, but monocytes and neutrophils as well. Our previous observations that monocyte depletion using clodronate-laden liposomes prior to induction of HIT in a passive immunization model mitigated the prothrombotic state, but paradoxically exacerbated initial thrombocytopenia, suggest that our understanding of the relationship between monocyte activation, platelet activation, thrombocytopenia and thrombosis is unsettled. Chondroitin sulfate is the predominant GAG expressed by platelets; therefore, they bind PF4 less avidly than monocytes, which have a cell surface rich in heparan and dermatan sulfates. Based on this cell-type difference in surface affinity for PF4, we hypothesized that: 1) monocytes and perhaps other vascular cells bind PF4 and form surface PF4/HIT antigenic complexes preferentially when compared with platelets, and 2) depletion of this high-affinity (monocyte) “sink” shifts PF4 binding to platelets making them more targeted. Flow cytometric analysis of whole mouse and human blood support these hypotheses as monocytes bind ∼100-fold more FITC-labeled human (h) PF4 than platelets or red blood cells and ∼10-fold more than neutrophils or lymphocytes. When isolated platelets and white blood cells are admixed, the amount of exogenously added hPF4 bound to platelets is inversely related to the leukocyte:platelet ratio. In vitro, exposure of whole blood to the HIT-like monoclonal antibody KKO plus recombinant hPF4 generated an intense platelet activation characterized by binding of annexin V and factor Xa, consistent with formation of coated platelets. Importantly, formation of coated platelets was attenuated by monocyte depletion from whole blood samples or by inhibition of thrombin by PPACK. We then infused KKO into transgenic mice expressing hPF4 and hFcgRIIA to induce HIT and followed the temporal profile of antibody binding to various cell types. Binding of KKO to monocytes was detected within 30 min of injection. These monocytes remained the predominant target over the first 4 hrs, after which binding decreased as the circulating monocytes were depleted. The mechanism and implications of this relatively late monocytopenia during HIT is under study. However, these data provide an explanation of how clodronate-laden monocyte depletion prior to inducing HIT exacerbated thrombocytopenia in the murine model of HIT, while decreasing the prothrombotic state: Early in the disease when PF4 is limited, monocytes selectively bind the PF4 and are targeted by HIT antibodies, which induces tissue factor and generates thrombin, but limits initial thrombocytopenia. Later, after induction of large amounts of TF, activated monocytes are cleared, shifting the target of antigen-antibody interactions to the surface of platelets, enhancing their response to available thrombin, thereby establishing a feed-forward prothrombotic cycle and more platelet clearance. In conclusion, we propose that HIT evolves from a monocyte-focused to a platelet-focused disease and that early intervention to prevent monocyte activation provides a new important potential therapeutic target for intervention at the earliest stages of disease recognition that may become less effective as time passes. Disclosures: Cines: Amgen Inc.: Consultancy; GlaxoSmithKline: Consultancy; Eisai: Consultancy.

Abstract 635 HIT is an immune mediated prothrombotic disorder often associated with life- and limb-threatening thrombosis caused by antibodies to a complex between platelet factor 4 (PF4) and heparin. Platelet activation and clearance are considered key to the pathogenesis. Direct thrombin inhibitors, the most commonly used anticoagulant therapy in the treatment of HIT, provide incomplete prevention against development of new thrombi and little effect on the incidence of loss of limb and life. Thus, there is a need for a better understanding of pathogenesis of HIT and new approaches to therapy. We and others have shown that HIT not only is associated with platelet activation, but also involves activation of monocytes and endothelial cells, which together increase thrombin generation that may affect both the amount and structural properties of the resultant fibrin clot. However, this proposed increase in fibrin formation though suspected, has never been directly investigated. Previously, we have shown in the cremaster muscle laser-injury model of thrombosis induced by the HIT-like murine monoclonal anti-hPF4/heparin antibody KKO that transgenic mice expressing both human PF4 (hPF4+) and hFcγRIIA developed larger, more fibrin-rich occlusive thrombi than in control mice expressing hPF4 or hFcγRIIA alone. To quantify fibrin formation in a more controlled setting, we simulated HIT in a BioFlux microfluidic channel system coated with von Willebrand factor by perfusing whole blood at a venule shear stress of 20 dyne/cm2 at 37°C for 10 min. Platelets were labeled by adding Calcein-AM (3 μM) and fibrin was visualized by adding Alexa 647 labeled fibrinogen (1.5 μg/ml) to the whole blood prior to the perfusion. Using NaCitrate-anticoagulated human blood, we observed that recalcified human blood samples exposed to KKO plus hPF4 formed large platelet thrombi and an extensive fibrin network, with fibers radiating from the platelet aggregates and often organized along the direction of flow. In contrast, control samples exposed to a combination of the non-pathogenic anti-hPF4 monoclonal antibody RTO plus hPF4 showed little fibrin and less organization. Quantitative fluorescence analysis showed nine times more fibrin formed after stimulation with KKO plus PF4 than RTO plus PF4. Inhibition of KKO-mediated platelet activation by blocking FcgRIIA with Fab fragments of monoclonal antibody IV.3 in whole blood suppressed platelet adhesion by > 80%, but decreased fibrin formation by only ∼40%. On the other hand, addition of a selective inhibitor of the Syk tyrosine kinase PRT-060318 (Reilly et al., Blood 2011:117:2241–6; kindly provided by Dr. Uma Sinha, Portola Pharmaceuticals) to whole blood at a concentration of 3 μM suppressed both platelet adhesion and fibrin formation by 80% and 70%, respectively. IV.3 inhibits platelet activation alone, while we have shown that PRT-060318 inhibits both platelet activation and monocyte activation with the subsequent release of tissue factor-rich microparticles. These results provide a mechanistic basis for the use of novel therapies in HIT such as fibrinolytic agents. To do so, we studied a novel chimeric pro-fibrinolytic composed of a C-terminal thrombin-specific activatable low molecular weight urokinase (uPA-T) that has its plasmin-activation site replaced by a thrombin cleavage site and linked at its N-terminus to a single-chain variable region (scFv) that binds with high affinity to human platelet aIIb, designed to deliver the agent to sites of incipient thrombosis. Preliminary results show that uPA-T profoundly suppressed fibrin accumulation in both in vitro and in an in vivo model of HIT. This novel approach to therapy takes advantage of our growing understanding of the pathogenesis of the prothrombotic nature of HIT including monocyte activation and formation of fibrin-rich clots. Such therapeutics may be especially effective as targeted therapy in HIT. Disclosures: Cines: Amgen Inc.: Consultancy; GlaxoSmithKline: Consultancy; Eisai: Consultancy.

Abstract 191 Outside-in signal transduction is one of several autocrine amplification loops that platelets employ to stabilize and consolidate a platelet thrombus following their adhesion to each other or to components of the extracellular matrix. Binding of soluble fibrinogen to activated integrin αIIbβ3 on the platelet surface, or binding of αIIbβ3 to platelet-immobilized fibrinogen, initiates an outside-in signaling cascade that results in the activation of integrin β3-associated Src family kinases, which in turn phosphorylate tyrosine residues within the cytoplasmic domain of the immunoreceptor tyrosine-based activation motif (ITAM)-containing adaptor protein, FcγRIIa. “Activation” of FcγRIIa sets off a cascade of events that result in the assembly and activation of other key signaling intermediates, including the tyrosine kinase Syk and phospholipase Cγ2(PLCγ2), through its lipase activity, generates lipid products that support a multitude of cellular activation responses, including cytoskeletal rearrangements leading to platelet shape change and spreading, secretion of platelet granules, and activation of additional cell surface integrins. We have previously shown that either antibody-mediated or genetic disruption of the functional interaction between integrin αIIbβ3 and FcγRIIa blocks tyrosine phosphorylation of FcγRIIa, Syk, and PLCγ2, and inhibits platelet spreading on immobilized fibrinogen. The physiological significance of FcγRIIa in supporting platelet thrombus formation, however, remains unknown. To further explore the importance of FcγRIIa in platelet function, we compared the relative ability of wild-type FcγRIIa-negative and transgenic FcγRIIa-positive (FcγRIIaTGN) murine platelets to support thrombosis and hemostasis in a number of well-accepted models of platelet function. FcγRIIaTGN platelets exhibited increased tyrosine phosphorylation of Syk and PLCγ2 and increased spreading upon interaction with immobilized fibrinogen. FcγRIIaTGN platelets also retracted a fibrin clot faster than did wild-type FcγRIIa-negative platelets. When anti-coagulated whole blood was perfused over a collagen-coated flow chamber under conditions of arterial shear, the rate and extent of adhesion, aggregation, and thrombus formation was significantly increased for FcγRIIaTGN platelets compared to their wild-type murine counterparts. Addition of Fab fragments specific for FcγRIIa to whole blood derived from either humans or FcγRIIaTGN mice strongly inhibited thrombus formation in the arterial in vitro flow chamber assay. Finally, to examine the in vivo relevance of FcγRIIa, mice were subjected to two models of vascular injury: electrolytic injury of the femoral vein and laser injury of cremaster arterioles. In both in vivo models, FcγRIIaTGN mice displayed increased thrombus formation compared with their wild-type, FcγRIIa-negative counterparts. Taken together, these data establish FcγRIIa as a physiologically-important functional conduit for αIIbβ3–mediated outside-in signaling, and suggest that modulating the activity of this novel integrin/ITAM pair might be effective in controlling thrombosis. Disclosures: No relevant conflicts of interest to declare.

Abstract 536 HIT is an iatrogenic, prothrombotic, immune-mediated disorder associated with antibodies directed against platelet factor 4 (PF4) and heparin complexes. Once these pathogenic anti-PF4/heparin antibodies develop, they recognize and bind to antigenic complexes formed on cell surfaces composed of PF4 released from platelets bound to cell membrane glycosaminoglycans (GAGs). Unlike platelets, which have only chondroitin sulfate on their surface, endothelial cells express mainly heparan sulfate resulting in a very high capacity to form PF4/GAG complexes and become targets for HIT antibodies. In fact, activation of endothelial cells by HIT antibodies was recognized almost 25 years ago (Cines et. al., 1987). We now show direct interaction of the HIT-like monoclonal antibody KKO with perithrombus endothelial cells, which propagates thrombosis following laser induced cremaster vessel injury in a passive immunization murine model of HIT. PF4 released from activated platelets at the site of laser injury binds rapidly and predominantly to the adjacent endothelium and is recognized by KKO in both transgenic mice expressing only human PF4 (hPF4+) and mice expressing hPF4 and human FcgRIIA on platelets (hPF4+/FcgRIIA+). These studies show that HIT-like antigenic complexes are present within thrombi prior to antibody exposure, predominantly localized to endothelial cells, which is consistent with their surface GAGs having higher affinity for PF4 than platelets. These endothelial cell surface PF4/GAG complexes, and with it KKO binding, are dissociated by high concentrations of intravenous heparin. Infusion of KKO in hPF4+/FcgRIIA+ mice but not in control mice, reinitiates growth of previously stable thrombi at sites of laser injury, leading to a significant increase in vascular occlusion (24/33 injuries, 73%, compared to 0/30 in control mice; P Disclosures: No relevant conflicts of interest to declare.