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SummaryEmerging evidence supports a role for platelets in the progression of atherosclerosis in addition to an involvement in thrombotic vascular occlusion. Platelet Factor 4 (PF4), a chemokine released by activated platelets, stimulates several pro-atherogenic processes. Therefore, we examined the localization of PF4 and the homologous protein, Neutrophil Activating Protein-2 (NAP-2) in lesions representing the evolution of human atherosclerotic plaques. Carotid plaques from 132 patients with critical carotid stenosis and 6 autopsy specimens were studied. Clinical, histologic and immunohistochemical data were analyzed using a χ2-test. PF4 was detected in the cytoplasm of luminal and neovascular endothelium, in macrophages and in regions of plaque calcification. The presence of PF4 in macrophages and neovascular endothelium correlated with lesion grade (p = 0.004; p = 0.044). Staining of macrophages for PF4 correlated with the presence of symptomatic atherosclerotic disease (p = 0.028). In early lesions, PF4 was commonly found in macrophages of early lesions (Grade I/II), whereas NAP-2 was rarely present.In conclusion, correlation between PF4 deposition, lesion severity and symptomatic atherosclerosis suggests that persistent platelet activation may contribute to the evolution of athero-sclerotic vascular lesions. These studies support the rationale for the chronic use of anti-platelet therapy in patients at risk for developing symptomatic atherosclerosis.

The structural basis of the interaction between single-chain urokinase-type plasminogen activator (scuPA) and its receptor (uPAR) is incompletely defined. Several observations indicated the kringle facilitates the binding of uPA to uPAR. A scuPA variant lacking the kringle (ΔK-scuPA) bound to soluble uPAR (suPAR) with the similar “on-rate” but with a faster “off-rate” than wild-type (WT)-scuPA. Binding of ΔK-scuPA, but not WT-scuPA, to suPAR was comparably inhibited by its growth factor domain (GFD) and amino-terminal fragment (ATF). ATF and WT-scuPA, but not GFD, scuPA lacking the GFD (ΔGFD-scuPA), or ΔK-scuPA reconstituted the isolated domains of uPAR. ATF completely inhibited the enzymatic activity of WT-scuPA-suPAR unlike comparable concentrations of GFD. Variants containing mutations that alter the charge, length, or flexibility of linker sequence (residues 43-49) between the GFD and the kringle displayed a lower affinity for uPAR, were unable to reconstitute uPAR domains, and their binding to uPAR was inhibited by GFD in the same manner as ΔK-scuPA. A scuPA variant in which the charged amino acids in the heparin binding site (HBS) in the kringle domain were mutated to alanines behaved like ΔK-scuPA, indicating that that the structure of the kringle as well as its interaction with the GFD govern receptor binding. These data demonstrate an important role for the kringle in stabilizing the binding of scuPA to uPAR. (Blood. 2003;102:3600-3608)

Heparin-induced thrombocytopenia and thrombosis (HIT/T) is a common immune-mediated disorder often manifested by life-threatening thrombosis. There is increasing evidence to indicate that HIT/T is caused by antibodies to complexes between platelet factor 4 (PF4) and heparin that activate platelets, monocytes and vascular endothelium leading to the generation of thrombin. Advances in defining the immunological basis of HIT/T have yielded insights into the antigenic determinants, antibody-antigen interactions and effector responses that contribute to its pathogenesis. However, these studies also reveal that anti-PF4/heparin antibodies develop far more commonly than clinically overt disease, raising questions as to serologic and other factors that predispose to clinical thrombocytopenia and thrombosis. An improved understanding of the natural history of HIT/T and the introduction of alternative anticoagulants have led to a somewhat improved clinical outcome. The recent development of a monoclonal anti-heparin/PF4 antibody and the establishment of a murine model of HIT/T may help to better define the pathogenesis and management of this common autoimmune disorder.

Summary form only given. Tissue plasminogen activator (tPA) triggers plasmin generation and subsequent blood clot dissolution. However, its therapeutic use is limited by an extremely short blood half-life. Previous studies by our group have shown that the half-life of tPA can be prolonged considerably by employing erythrocytes as natural carriers of tPA and that RBC carriage of tPA facilitates fibrinolysis of the nascent clot. In the present work we characterized the lysis of the nascent clots by RBC-tPA complex under flow conditions using an original in vitro model. Normal human RBC or RBC conjugated with activase (ACT, wild-type tPA) were entrapped in nascent plasma clots formed under venous flow conditions (62.5 s/sup -1/). Video-microscopy was used to compare formation and lysis of both RBC and RBC-tPA containing clots in separate flow chambers. Both types of clots displayed similar clot formation time (10-15 min). However, while clots containing normal RBC did not degrade even after a 90 min perfusion with buffer, degradation of the clots containing RBC-tPA was observed within 15 min. Lysis quantification with radiolabelled fibrinogen demonstrated that a 40 min perfusion of buffer over the RBC-tPA clot reduced its fibrin content and red cell content by 70% and 85%, respectively. In contrast, no changes in fibrin and red cell count were detected in the control clot. These data support the notion that RBC carriage of plasminogen activators may be useful for thromboprophylaxis.

Arterial occlusive disorders are a leading cause of human morbidity. We hypothesized that ectopic expression of fibrinolytic proteins in platelets could be used to favorably alter the hemostatic balance at sites of thrombosis. To test our hypothesis, we directed murine urokinase-type plasminogen activator transgene expression to platelets using a platelet factor 4 promoter. Urokinase was selectively expressed and stored in the platelets of these mice. These transgenic mice had altered platelet biology and a bleeding diathesis similar to that seen in patients with Quebec platelet disorder, affirming the role of ectopic urokinase expression as the etiology of this inherited disease. These mice were resistant to the development of occlusive carotid artery thrombosis in the absence of systemic fibrinolysis and displayed rapid resolution of pulmonary emboli. Moreover, transfusion of urokinase-expressing platelets into wild-type mice prevented formation of occlusive arterial thrombi. These studies show the feasibility of delivering fibrinolytic agents to sites of incipient thrombus formation through selective storage in platelets and offer a new strategy to prevent thrombosis and hemorrhage.

Drug targeting to a highly expressed, noninternalizable determinant up-regulated on the perturbed endothelium may help to manage inflammation and thrombosis. We tested whether inter-cellular adhesion molecule-1 (ICAM-1) targeting is suitable to deliver antithrombotic drugs to the pulmonary vascular lumen. ICAM-1 antibodies bind to the surface of endothelial cells in culture, in perfused lungs, and in vivo. Proinflammatory cytokines enhance anti-ICAM binding to the endothelium without inducing internalization. 125I-labeled anti-ICAM and a reporter enzyme (β-Gal) conjugated to anti-ICAM bind to endothelium and accumulate in the lungs after intravenous administration in rats and mice. Anti-ICAM is seen to localize predominantly on the luminal surface of the pulmonary endothelium by electron microscopy. We studied the pharmacological effect of ICAM-directed targeting of tissue-type plasminogen activator (tPA). Anti-ICAM/tPA, but not control IgG/tPA, conjugate accumulates in the rat lungs, where it exerts plasminogen activator activity and dissolves fibrin microemboli. Therefore, ICAM may serve as a target for drug delivery to endothelium, for example, for pulmonary thromboprophylaxis. Enhanced drug delivery to sites of inflammation and the potential anti-inflammatory effect of blocking ICAM-1 may enhance the benefit of this targeting strategy.

In this issue of Blood , Langhauser and colleagues report that kininogen knockout mice (KNG −/− ) are protected from stroke induced by transient mechanical occlusion of the middle artery (MCAO). 1 KNG −/− mice develop smaller infarcts, less neurologic impairment, and exhibit lower mortality than wild-type (WT) mice studied at 24 hours and beyond. Benefit was retained in elderly mice and was conferred through reduction in microvascular thrombosis, preservation of blood-brain-barrier function, and attenuation of inflammation. These are important findings because they provide insight into the pathophysiology of stroke and identify potential novel targets for intervention.

Although idiopathic thrombocytopenic purpura (ITP) is the most common autoimmune hematologic disorder, little is known about the associated autoantibodies on a molecular level. Consequently, diagnostic assays and therapy for ITP lack specificity. To avoid technical limitations imposed by B-cell immortalization methods, we used repertoire cloning (Fab/phage display) to clone platelet autoantibodies and examine the relation between immunoglobulin (Ig) gene usage, clonality, and antigen specificity. Phage display libraries were constructed from splenocytes from 2 patients with chronic ITP, and competitive cell-surface selection was used to isolate several dozen unique IgG platelet-specific autoantibodies. Platelet-reactive Fabs in both patients were associated almost exclusively with rearrangements of a single Ig heavy-chain variable-region gene (V(H)3-30), despite an apparent diversity of antigen specificities. Comparative analysis of platelet-reactive Fab Ig gene rearrangements from each patient suggested that they evolved from a restricted number of B-cell clones through somatic mutation with high replacement-to-silent mutation ratios. Although V(H)3-30-encoded heavy chains were found with light chains encoded by several different Ig genes, molecular repairing experiments showed exquisite restriction on the specific heavy- and light-chain pairings that permitted platelet reactivity. Together, these data suggest that the development of platelet-reactive antibodies associated with ITP is driven by an encounter with diverse platelet antigens through the clonal expansion of B cells using genetically restricted and highly specific combinations of heavy- and light-chain gene products. The extraordinarily high usage of the V(H)3-30 heavy-chain gene in these patients has implications for the pathogenesis, diagnosis, and management of chronic ITP.

Using tracings of125I-labeled fibrin(ogen) in rodents, we examined the hypothesis that platelets impede the lysis of pulmonary emboli.125I-Microemboli (ME, 3–10 micron diameter) lodged homogeneously throughout the lungs after intravenous injection in both rats and mice (60% of injected dose), caused no lethality, and underwent spontaneous dissolution (50 and 100% within 1 and 5 h, respectively). Although lung homogenates displayed the most intense fibrinolytic activity of all the major organs, dissolution of ME was much slower in isolated perfused lungs (IPL) than was observed in vivo. Addition of rat plasma to the perfusate facilitated ME dissolution in IPL to a greater extent than did addition of tissue-type plasminogen activator alone, suggesting that permeation of the clot by plasminogen is the rate-limited step in lysis. Platelet-containing ME injected in rats lysed much more slowly than did ME formed from fibrin alone.125I-Thrombi, formed in the pulmonary vasculature of mice in response to intravascular activation of platelets by injection of collagen and epinephrine, were essentially resistant to spontaneous dissolution. Moreover, injection of the antiplatelet glycoprotein IIb/IIIa antibody 7E3 F(ab′)2facilitated spontaneous dissolution of pulmonary ME and augmented fibrinolysis by a marginally effective dose of Retavase (10 μg/kg) in rats. These studies show that platelets suppress pulmonary fibrinolysis. The mechanism(s) by which platelets stabilize ME and utility of platelet inhibitors to facilitate their dissolution deserves further study.

Heparin-induced thrombocytopenia/thrombosis (HIT/T) is a common complication of heparin therapy that is caused by antibodies to platelet factor 4 (PF4) complexed with heparin. The immune response is polyclonal and polyspecific, ie, more than one neoepitope on PF4 is recognized by HIT/T antibodies. One such epitope has been previously identified; it involves the domain between the third and fourth cysteine residues in PF4 (site 1). However, the binding sites for other HIT/T antibodies remain to be defined. To explore this issue, the binding site of KKO, an HIT/T-like murine monoclonal antibody, was defined. KKO shares a binding site with many HIT/T antibodies on PF4/heparin, but does not bind to site 1 or recognize mouse PF4/heparin. Therefore, the binding of KKO to a series of mouse/human PF4 chimeras complexed with heparin was examined. KKO recognizes a site that requires both the N terminus of PF4 and Pro34, which immediately precedes the third cysteine. Both regions lie on the surface of the PF4 tetramer in sufficient proximity (within 0.74 nm) to form a contiguous antigenic determinant. The 10 of 14 HIT/T sera that require the N terminus of PF4 for antigen recognition also require Pro34 to bind. This epitope, termed site 2, lies adjacent to site 1 in the crystal structure of the PF4 tetramer. Yet sites 1 and 2 can be recognized by distinct populations of antibodies. These studies further help to define a portion of the PF4 tetramer to which self-reactive antibodies develop in patients exposed to heparin.

False normal von Willebrand factor activity by monoclonal antibody-based ELISA in a patient with type 2A(IID) von Willebrand disease

Background . Studies have demonstrated a high incidence of antibodies to heparin/platelet factor 4 complexes, the antigen in heparin-induced thrombocytopenia, in patients after cardiopulmonary bypass surgery. In many hospitals, beef lung heparin has been used historically for cardiopulmonary bypass, and there has been reluctance to change to porcine heparin despite concerns of an increased incidence of heparin-induced thrombocytopenia in patients receiving bovine heparin. Methods . A prospective randomized trial comparing bovine and porcine heparin in cardiopulmonary bypass surgery was conducted. Presurgery and postsurgery heparin antibody formation was studied using the serotonin release assay and a heparin/platelet factor 4 enzyme-linked immunosorbent assay. Results . Data available on 98 patients, randomized to receive either bovine or porcine heparin, revealed no significant difference in patient positivity by serotonin release assay (12% in both groups) or by the heparin/platelet factor 4 enzyme-linked immunosorbent assay (29% with porcine and 35% with bovine heparin) postoperatively. There were no significant differences between preoperative and postoperative platelet counts or thromboembolic complications. Conclusions . Our study does not support the belief that bovine heparin is more likely than porcine heparin to induce the development of antibodies to heparin/platelet factor 4.

Publisher Summary Vascular inflammation affects the progression of atherosclerosis through diverse mechanisms. One pathway may involve the enhancement of lipoprotein retention. It has been estimated that 30-50% of the risk of symptomatic coronary artery disease involves factors that complement hypercholesterolemia and other identified abnormalities of lipid metabolism. Part of this variability appears to result from factors that act locally to alter lipoprotein metabolism. The rate of lipoprotein transport into vessel walls considerably exceeds their rate of accumulation. This indicates that retention is the rate-limiting step in lipoprotein accumulation. Retained lipoproteins are subject to oxidation, aggregation and other modifications that promote lipid accumulation and vascular injury. Consistent with this idea is the finding that reduction of vascular retention time may help to prevent oxidant-induced vascular injury. Inflammation also contributes to lesion progression by activating the coagulation system. There is extensive literature demonstrating the presence of fibrin both in native atherosclerotic lesions and in the accelerated atherosclerosis that develops in allografts.

Binding of urokinase-type plasminogen activator (uPA) to its receptor (uPAR/CD87) regulates cellular adhesion, migration, and tumor cell invasion. However, it is unclear how glycosyl phosphatidylinositol-anchored uPAR, which lacks a transmembrane structure, mediates signal transduction. It has been proposed that uPAR forms cis-interactions with integrins as an associated protein and thereby transduces proliferative or migratory signals to cells upon binding of uPA. We provide evidence that soluble uPAR (suPAR) specifically binds to integrins alpha4beta1, alpha6beta1, alpha9beta1, and alphavbeta3 on Chinese hamster ovary cells in a cation-dependent manner. Anti-integrin and anti-uPAR antibodies effectively block binding of suPAR to these integrins. Binding of suPAR to alpha4beta1 and alphavbeta3 is blocked by known soluble ligands and by the integrin mutations that inhibit ligand binding. These results suggest that uPAR is an integrin ligand rather than, or in addition to, an integrin-associated protein. In addition, we demonstrate that glycosyl phosphatidylinositol-anchored uPAR on the cell surface specifically binds to integrins on the apposing cells, suggesting that uPAR-integrin interaction may mediate cell-cell interaction (trans-interaction). These previously unrecognized uPAR-integrin interactions may allow uPAR to transduce signals through the engaged integrin without a hypothetical transmembrane adapter and may provide a potential therapeutic target for control of inflammation and cancer.

Understanding the basic forces that determine molecular recognition helps to elucidate mechanisms of biological processes and facilitates discovery of innovative biotechnological methods and materials for therapeutics, diagnostics, and separation science. The ability to measure interaction properties of biological macromolecules quantitatively across a wide range of affinity, size, and purity is a growing need of studies aimed at characterizing biomolecular interactions and the structural elements that drive them. Optical biosensors have provided an increasingly impactful technology for such biomolecular interaction analyses. These biosensors record the binding and dissociation of macromolecules in real time by transducing the accumulation of mass of an analyte molecule at the sensor surface coated with ligand molecule into an optical signal. Interactions of analytes and ligands can be analyzed at a microscale and without the need to label either interactant. Sensors enable the detection of bimolecular interaction as well as multimolecular assembly. Most notably, the method is quantitative and kinetic, enabling determination of both steady-state and dynamic parameters of interaction. This article describes the basic methodology of optical biosensors and presents several examples of its use to investigate such biomolecular systems as cytokine growth factor–receptor recognition, coagulation factor assembly, and virus–cell docking.

Proteins linked to cell membranes by a glycosylphosphatidylinositol (GPI) anchor must first undergo cleavage by a putative transamidase between the omega and omega + 1 positions within a proposed small amino acid (SAD) domain in the carboxy terminus of the nascent polypeptide. The requirements for such processing, defined in an engineered placental alkaline phosphatase construct (miniPLAP), suggest the SAD domain functions as an autonomous unit within the context of an otherwise permissive carboxy-terminal sequence with only certain amino acids tolerated at the omega, omega + 1, and omega + 2 positions. To test whether this hypothesis could be generalized, we engineered a chimeric molecule containing the extracellular domain of miniPLAP and the carboxy-terminal portion of the urokinase receptor (MP/uPAR) into which various amino acid substitutions were introduced. The variant proteins were translated and metabolically labeled in vitro using a cell-free translation system that contains the enzymatic machinery required for carboxy-terminal processing and GPI anchor addition. The results of this study indicate that the SAD domain functions as an independent, but not an autonomous, unit. The requirements for processing in miniPLAP and MP/uPAR differed markedly in some respects, in part due to the influence of the amino acid at the omega + 4 position which both modified cleavage between the omega and omega + 1 positions and permitted a second cleavage site to be generated in some cases. In addition, substitution of bulky hydrophobic amino acids in series at the omega + 2 and omega + 3 positions inhibited carboxy-terminal processing in a dose-dependent manner, suggesting the presence of a critical docking site adjacent to the cleavage site. These results suggest the carboxy-terminal transamidase recognizes a more extended structure similar to the mechanism proposed for serine proteases. Further, the data provide a potential means for isolating the transamidase.

Platelets play a vital role in hemostasis. Until recently, there was no means to increase platelet production in order to treat or prevent bleeding. In this issue of Blood , Jenkins and colleagues describe the results of a phase 1 clinical trial in healthy adults with eltrombopag, a first-in-class

Single-chain urokinase plasminogen activator (scuPA), the unique form secreted by cells, expresses little intrinsic plasminogen activator activity. scuPA can be activated by proteolytic cleavage to form a two-chain enzyme (tcuPA), which is susceptible to inhibition by plasminogen activator inhibitor type I (PAI-1). scuPA is also activated when it binds to its cellular receptor (uPAR), in which case the protein remains as a single chain molecule with less susceptibility to PAIs. Fibrin clots are invested with PAI-1 derived from plasma and from activated platelets. Therefore, we compared the fibrinolytic activity of complexes between scuPA and recombinant soluble uPAR (suPAR) to that of scuPA, tcuPA, and tcuPA/suPAR complexes. scuPA/suPAR complexes mediated the lysis of plasma-derived fibrin clots 14-fold more extensively than did equimolar concentrations of scuPA and threefold more extensively than did tcuPA or tcuPA/suPAR, respectively. The enhanced catalytic activity of scuPA/suPAR required that all three domains of the receptor be present, correlated with its PAI-1 resistance, was not dependent on fibrin alone, and required a plasma cofactor that was identified as IgG. Human IgG bound specifically to suPAR and scuPA/suPAR as determined by using affinity chromatography and immunoprecipitation. Plasma depleted of IgG lost most of its capacity to promote the fibrinolytic activity of scuPA/suPAR, and the activity of the complex was restored by adding plasma concentrations of purified IgG. These studies indicate that scuPA/suPAR can function as a plasminogen activator in a physiological milieu.© 1998 by The American Society of Hematology.

Purpose: The association of anti-Br a immunoglobulin G (IgG) platelet alloantibodies with the development of thrombocytopenia in neonates of mothers with autoimmune thrombocytopenic purpura (ITP) is reported. Methods: Between March 1994 and July 1997, 28 consecutive pregnant women with ITP seen at New York Hospital were screened for platelet-reactive antiglycoprotein (glycoprotein [GP] IIb/IIIa, Ib/IX, and Ia/IIa) antibodies. Results: The sera from 6 of these 28 women contained IgG alloantibodies to GP Ia/IIa directed against the Br a (HPA-5b) antigen. Only three families each had at least one Br a + and at least one Br a - infant. Platelet typing in these families revealed that the mothers were Br b/b (Br a -) and the fathers were Br a/b (Br a +). Platelet counts

Bennett and colleagues report that approximately one third of children and adolescents with severe or refractory chronic immune thrombocytopenic purpura (ITP) showed a sustained response to treatment with the monoclonal anti-C20 antibody, rituximab. This prospective study is notable for several

The internalization and degradation of cell-associated urokinase type plasminogen activator (uPA) through the alpha2-macroglobulin receptor/low density lipoprotein-related receptor (alpha2MR/LRP) represent important steps in the control of plasmin formation. Complexes between two chain urokinase (tcuPA) and plasminogen activator type 1 are degraded rapidly whereas single chain urokinase (scuPA) is not, suggesting that alpha2MR/LRP requires specific epitopes in the serpin for effective function. We report an alternative mechanism that may contribute to this process. The binding of scuPA to LM-TK- cells that lack the uPA receptor was stimulated by the hexapeptide EEIIMD, corresponding to amino acids 350-355 of plasminogen activator type 1, which contacts the sequence RHRGGS, corresponding to amino acids 179-184 in uPA. EEIIMD increased the Bmax of scuPA binding 4-fold with the half-maximal effect achieved at a peptide concentration of 50 microM. Stimulation was dependent on the charge on the COOH-terminal amino acid but not on the NH2 terminus of the peptide. EEIIMD also stimulated the internalization and degradation of scuPA. Both the binding and internalization of scuPA in the presence of EEIIMD were blocked by recombinant, 39-kDa alpha2MR/LRP-associated protein as well as by an anti-alpha2MR/LRP antibody. EEIIMD also stimulated the binding of scuPA to purified alpha2MR/LRP. EEIIMD had no effect on the binding of tcuPA or of complexes between scuPA and its receptor. These results suggest that EEIIMD regulates the binding of scuPA with alpha2MR/LRP. These findings also suggest a potential mechanism by which scuPA can be cleared which is independent of activation by plasmin or binding to uPA receptor.

Background Patients with cardiovascular disease almost invariably receive heparin before cardiopulmonary bypass surgery, which places them at risk of developing heparin-associated antibodies with a risk of thromboembolic complications. This study was designed to determine the prevalence of heparin-induced antibodies in patients before and after cardiopulmonary bypass surgery. Methods and Results Plasma from 111 patients was tested before surgery and 5 days after surgery for heparin-dependent platelet-reactive antibodies with a 14 C-serotonin–release assay (SRA) and for antibodies to heparin/platelet factor 4 complexes with an ELISA. Heparin exposure after surgery was minimized. Heparin-dependent antibodies were detected before surgery in 5% of patients with SRA and 19% of patients with ELISA. By the fifth postoperative day, there was a marked increase in patients positive on the SRA or ELISA (13% and 51%, respectively; P P =.017) and a positive ELISA (68%; P =.054) or SRA (30%; P =.002) after surgery. However, there was no difference in the prevalence of thrombocytopenia or thromboembolic events between the antibody-positive and -negative groups. Conclusions Approximately one fifth of patients undergoing cardiopulmonary bypass surgery have heparin-induced platelet antibodies detectable before the procedure as a result of prior heparin exposure, and many more develop antibodies after surgery. The absence of an association between these antibodies and thromboembolic complications in this study may be, in part, attributable to careful avoidance of heparin after surgery. The high prevalence of heparin-induced antibodies in this setting suggests that these patients may be at risk of developing thrombotic complications with additional heparin exposure.

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.

HIT is an iatrogenic complication of heparin therapy caused by antibodies that recognize the platelet chemokine, platelet factor 4 (PF4), complexed to heparin or to cellular glycosaminoglycans (GAG). Unlike most other immune thrombocytopenias, HIT is markedly prothrombotic. We have proposed that this prothrombotic tendency is due to binding of pathogenic antibodies to PF4 complexes attached to the surface GAGs expressed by all intravascular cells, including platelets with their relatively low affinity surface GAGs, chondroitin sulfates, and monocytes with their higher affinity membrane GAGs, heparan and dermatan sulfates. Using isolated monocytes from healthy volunteers, we show by scanning electron microscopy that the addition of 10-50 µg/ml of recombinant human PF4 causes the appearance of ∼200 nm “knobs” on the cell surface. Subsequent addition of a HIT-like monoclonal antibody KKO at 50 µg/ml to the PF4-coated cells markedly alters their surface with the appearance of larger, up to 1-2 µm, membrane “blebs”. These blebs increase in size over time (15-60 minutes) and are shed from the cells. After shedding of these blebs, the monocytes lose their typical ruffled surface and appear spherical. These surface changes in the presence of KKO and PF4 are not seen in the presence of PF4 and 50 µg/ml of the anti-PF4 monoclonal antibody RTO, which does not induce the prothrombotic state of HIT. Platelets in suspension exposed to PF4 and KKO show by scanning electron microscopy similar knobs on their surface, but only minimally form blebs or microvesiculate. Platelets spread on fibrinogen in culture medium stimulated with PF4 and KKO and observed by hopping probe ion conductance microscopy progressively developed surface protrusions over a period of an hour, becoming more spherical. This morphological change was also observed in platelets exposed to IgG purified from 5 patients with HIT, but not when the KKO Fab fragment was tested. Neither PF4 alone nor with RTO antibody induced this morphological transition. Exposure to KKO plus PF4 for an hour induced minimal microvesiculation of platelets as measured by flow cytometry. Platelets adherent to fibrinogen underwent a similar morphological transition and did not microvesiculate after adding 50 mM of a thrombin-receptor activating peptide, whereas ADP-stimulated platelets rapidly microvesiculated during the same period of time. We believe that the “knobs” observed on monocytes and platelets represent aggregates of PF4-GAG complexes that are the targets of HIT antibodies. Bleb formation on monocytes and morphological transition of platelets result from clustering of knobs caused by bivalent HIT antibodies which cross-link Fc receptors. These blebs are released from monocytes, potentially becoming the microparticles found in the plasma of patients with HIT. In contrast, platelets treated with KKO plus PF4 showed minimal microvesiculation. This finding differs from reported high platelet microparticle counts in the plasma of HIT patients, suggesting that additional factors may be required to induce platelets to microvesiculate. Our images represent the first visualization of surface events when platelets and monocytes assume an active prothrombotic state. Whether these are unique to HIT or have wider applicability to the changes that occur in other prothrombotic, proinflammatory states needs to be addressed. 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.

The objective of this study was to determine whether cultured human trophoblasts migrate in response to changes in oxygen tension or temperature. Human trophoblastic cells distributed homogenously within individual wells of standard culture plates were subjected to oxygen and thermal gradients. The redistribution of cells was determined 90 min to 18 h after these gradients had been established. Trophoblastic cells did not migrate in response to gradients of oxygen or carbon dioxide applied in this manner. In contrast, the cells migrated in response to thermal gradients of less than 1 degree C in the direction of the warmer temperature. The response began within minutes, was reversed by a change in the direction of the thermal gradient, and was inhibited at high cell concentrations. Migration was independent of proliferation or protein synthesis, but required microfilament assembly. The capacity of trophoblasts to migrate in response to small difference of temperature within the physiologic range may contribute to the initiation of placental development before contact with the maternal circulation has been established.

Defensins are naturally occurring antimicrobial peptides that may participate in host defense against microorganisms. We previously reported that the amino acid sequence of leukocyte defensins resembles the lysine-binding site in the kringles of plasminogen and that defensin inhibits fibrinolysis mediated by tissue-type plasminogen activator (tPA) and plasminogen. In the present paper we analyze the mechanisms of this inhibition. Defensin binds specifically to cultured human umbilical vein endothelial cells (HUVEC) (half-maximal binding = 3 microM) as well as to fibrin. At saturating concentrations (5-10 microM), defensin stimulates the maximum binding of plasminogen to HUVEC and to fibrin approximately 10-fold. However, defensin inhibits plasminogen binding to both surfaces at concentrations10 microM. Defensin also inhibits tPA and plasminogen-mediated fibrinolysis in a dose-dependent manner at all concentrations tested. Fibrinolysis is almost totally inhibited by 6 microM defensin, a concentration that stimulates the binding of plasminogen to fibrin. Discordance between the enhancement of plasminogen binding and its activation cannot be explained by an inhibitory effect of defensin on tPA binding nor by inhibition of plasmin activity, each of which occur only at higher concentrations. Rather, these results suggest that plasminogen bound to fibrin in the presence of defensin is less susceptible to activation by tPA.

The purpose of the study was to attempt to distinguish pregnant women with gestational thrombocytopenia from those with idiopathic immune thrombocytopenia by eight different platelet antibody assays.Sera from pregnant women with presumed gestational thrombocytopenia (n = 160) and idiopathic immune thrombocytopenia (n=90) were prospectively tested for indirect and platelet-associated immunoglobulins G and M and complement C3, as well as for serotonin release. After the results were analyzed, a subset of patients were subsequently analyzed for circulating antiplatelet antibody directed against platelet membrane glycoprotein GPIIb/IIIa.Indirect immunoglobulin G was significantly greater in the 85 women with idiopathic immune thrombocytopenia than in the 129 women with gestational thrombocytopenia (p0.001). Platelet-associated immunoglobulin G was elevated in the majority of women, both those with gestational thrombocytopenia and those with idiopathic immune thrombocytopenia. There were also no statistically significant difference in the values for platelet-associated C3 or indirect immunoglobulin M and C3. Levels of platelet-associated immunoglobulin M showed a tendency to be higher in women with gestational thrombocytopenia (p=0.04), as did the values in the serotonin release assay (p=0.06).Our data demonstrate that patients with gestational thrombocytopenia had surprisingly high levels of platelet-associated immunoglobulin despite mild thrombocytopenia. Comparison of a relatively large number of patients with idiopathic immune thrombocytopenia and gestational thrombocytopenia indicates that women with idiopathic immune thrombocytopenia cannot be distinguished from those with gestational thrombocytopenia by means of one or more of the prototypic platelet antiglobulin tests currently in use. Our preliminary data with glycoprotein-specific assays indicate that they may be more useful.

Publisher Summary This chapter provides an overview of heparin-associated autoantibodies. The studies include intensive efforts to standardize the assay, define associations with disease or clinical presentation, and characterize antigenic and biological properties of these antibodies Heparin-induced thrombocytopenia (HIT) provides an interesting model of autoimmunity in which a heterologous mucopolysaccharide combines with a normal endogenous protein released in specific clinical settings to generate autoantibodies in susceptible, but immunologically ‘normal” individuals to cause thrombocytopenia and thrombosis. HIT antibodies cause normal platelets to aggregate and secrete serotonin in vitro. The serotonin release assay (SRA) provides a more objective endpoint than platelet aggregation studies that have variable sensitivity and specificity.

The diagnosis of heparin-induced thrombocytopenia (HIT) may be affirmed by demonstrating heparin-dependent anti-platelet antibodies using the 14C-serotonin release assay (SRA). In this study, results of the SRA was compared with the recently described platelet factor 4 (PF4)/heparin enzyme-linked immunosorbent assay (ELISA). Compared with the SRA, the sensitivity and specificity of a PF4/heparin ELISA was 87% and 92%, respectively, using an assay developed in our laboratory; and 90% and 98%, respectively, using a commercially developed kit (Diagnostica Stago, Asnieres, France). However, antibodies to PF4/heparin were also detected in up to 8% of patients whose plasma was negative by SRA, and 23% of patients receiving heparin who were not thrombocytopenic. These data indicate that results obtained with the PF4/heparin ELISA and the SRA are generally in accord in patients with a clinical diagnosis of HIT. However, discrepant results occur in approximately 20% of cases because of the greater sensitivity of ELISA and the possible involvement of other heparin-binding proteins. The fact that each assay contributes independent information in some cases must be considered in the sequence of test performance and in providing consultation to the practicing hematologist.

The pathogenesis of the antiphospholipid syndrome remains uncertain. Antibodies that react with phospholipids may not be directly responsible for cellular injury, but may be part of the immune network through which autoantibodies with pathogenic potential are generated. The latter may recognize proteins such as beta 2-glycoprotein I that form complexes with phospholipids, proteins whose functions depend upon interaction with phospholipids such as protein C and its cofactors, altered lipoproteins such as oxidized low-density lipoproteins, or other molecules that share only antigenic similarity. Thus, a spectrum of autoantibodies that recognize different lipid-protein complexes may develop in these patients and contribute to the observed clinical heterogeneity of the syndrome. Current techniques do not permit identification of the subset of patients with antiphospholipid antibodies at risk for thrombosis or abortion and there are no prospective, controlled trials addressing the prophylaxis or treatment of affected individuals. Identification of the cellular targets of antibodies to lipid-protein moieties is needed to identify patients at risk for these complications and as a means to monitor therapy.

Investigations mapped the region(s) on the light chain of high molecular weight kininogen (HK) that participates in cell binding. Sequential and overlapping peptides of domain 5 (D5H) were synthesized to determine its cell binding site(s). Three peptides from non-overlapping regions on D5H were found to inhibit biotin-HK binding to endothelial cells. Peptides GKE19 and HNL 21 weakly inhibited biotin-HK binding with IC50 of 792 and 215 microM, respectively. Peptide HKH20 inhibited biotin-HK binding with an IC50 of 0.2 microM. Two peptides, GGH18 and HVL24, which overlapped HKH20, also inhibited biotin-HK binding to endothelial cells with IC50 values of 108 and 0.8 microM, respectively. Biotinylated HKH20 directly bound to endothelial cells. HK and HKH20 bound at or near the same site on endothelial cells because HK inhibited biotin-HKH20 binding (IC50 = 0.2 microM). A polyclonal anti-HKH20 antibody also blocked biotin-HK binding. Peptides HKH20 and HVL24 and anti-HKH20 antibody also inhibited the procoagulant activity of plasma HK. These data indicated that the cell and artificial surface binding sites on D5H overlap. The orientation of HK on endothelial cells may be critical for the assembly and activation of contact system enzymes and the expression of kininogen's anti-thrombin activity.

Protease nexin-2/amyloid beta-protein precursor (PN-2/A beta PP) is a Kunitz-type protease inhibitor which has been shown to be a tight-binding inhibitor of enzymes, factors XIa and IXa (FIXa), suggesting a role for this protein in hemostasis. Since coagulant reactions are modulated on biologic surfaces, we investigated how 25:75 (mol/mol) phosphatidylserine/phosphatidylcholine vesicles (PSPC), thrombin-activated platelets, or umbilical vein endothelial cells influence inactivation of FIXa by PN-2/A beta PP. The Km of human or porcine FIXa activation of human factor X in the presence of PSPC, activated platelets, or endothelial cells in the absence or presence of thrombin-activated factor VIII (FVIIIa) was similar, (0.05-0.39 microM). The presence of FVIIIa increased the catalytic efficiency (kcat/Km ratio) of human and porcine factor IXa's activation of factor X 4952-406-fold, respectively. In the presence of PSPC, the Ki of human and porcine FIXa inhibition by PN-2/A beta PP was Ki = 1.9 x 10(-9) M and 5.8 x 10(-9) M, respectively. After the addition of FVIIIa to the reaction, the Ki for both human and porcine FIXa inhibition by PN-2/A beta PP on PSPC increased 13- and 4-fold to Ki = 2.5 x 10(-8) M and 2.4 x 10(-8) M, respectively. These Ki for inhibition of human FIXa on phospholipid vesicles by PN-2/A beta PP were similar when factor X activation was measured by chromogenic or activation peptide release assays. FVIIIa reduced the inhibition of FIXa by PN-2/A beta PP only in the presence of PSPC.(ABSTRACT TRUNCATED AT 250 WORDS)

The question, “Doctor, is it safe for me to become pregnant?” is one of the more charged issues confronting a woman with idiopathic thrombocytopenic purpura (ITP) and her physician. Maternal platelet counts can fall during even uncomplicated pregnancies. Some of the drugs commonly used to manage

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 2185 Introduction: ITP is characterized by low platelet counts due to increased platelet destruction and suboptimal platelet production. Romiplostim, which is approved for the treatment of chronic ITP in adults, is an Fc-peptide fusion protein (peptibody) that binds and activates the thrombopoietin receptor to stimulate platelet production. Results from a safety analysis of pooled data from ITP romiplostim clinical studies were previously reported (Rodeghiero et al, Haematologica 2010;95(s2) abstr 0184). Here we update the safety findings with the most inclusive clinical dataset of patients (pts) with ITP. Methods: Data from 13 studies of ITP were analyzed. Pts received romiplostim, placebo or medical standard of care (SOC) treatment at some time from July 2002 to June 2011. Data from the placebo/SOC arms were pooled. Results were adjusted for study duration and reported as rates per 100 patient-years (pt-yr) to reflect the unequal study time between pts given romiplostim and pts given placebo/SOC. In cases where pts enrolled in ≥ 1 study, data from the first and extension studies were combined. Data for pts who started on placebo/SOC and then received romiplostim were recorded as follows: data before pts received the first dose of romiplostim were included in the placebo/SOC group; data on or after the first dose of romiplostim were included in the romiplostim group regardless of any subsequent change in treatment. Results: Pts (N = 1,059) were mostly female (61%) and white (85%); 23 pediatric pts were included in the analysis. All pts had received prior ITP treatment per protocol entry criteria. Age, sex, race and prior ITP treatment were similar between groups. In studies where prior information on splenectomy was collected, 47% of pts had a splenectomy. Mean baseline platelet count: 21×109/L (standard deviation, 17×109/L). In total 921 pts received romiplostim, 65 received placebo/SOC, and 73 received placebo/SOC in the first study and romiplostim in subsequent studies. Mean weekly romiplostim dose: 4.2 μg/kg. Patient exposure to romiplostim: ≤ 1 yr, 47%; >1–2 yr, 26%; >2–3 yr, 15%; >3–4 yr, 6%; > 4 yr, 6%. Nineteen percent and 22% of pts who received romiplostim and placebo/SOC, respectively, did not complete participation in their first ITP study. Adverse events (AE) were reported in 94.2% and 93.5% of pts in the romiplostim and placebo/SOC groups, respectively. Most frequently reported AE (rates per 100 pt-yr) in the romiplostim vs placebo/SOC groups: headache (61 vs 58), contusion (48 vs 50) and epistaxis (35 vs 53). AE with > 10% difference in the romiplostim vs placebo/SOC groups: headache (36% vs 24%), arthralgia (24% vs 12%), nausea (20% vs 9%). Three pts developed neutralizing antibodies to romiplostim but did not develop neutralizing antibodies to thrombopoietin and did not become refractory to romiplostim. The Table summarizes AE. Conclusions: This integrated analysis of all available clinical studies to date involving the use of romiplostim in ITP provides long term safety information with some pts receiving romiplostim for over five yr. The AE profile was consistent with previously reported studies. Disclosures: Cines: GlaxoSmithKline: Consultancy; Amgen Inc.: Consultancy; Eisai: Consultancy. Gernsheimer:Amgen Inc.: Consultancy, Honoraria; Symphogen: Consultancy; Laboratorios Raffo SA: Honoraria; Clinical Options: Consultancy; Hemedicus Corporation: Honoraria; Glaxo-Smith Kline: Consultancy; Shionogi: Research Funding; Cangene: Consultancy. Wasser:Amgen Inc.: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Altomare:Amgen Inc.: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Wang:Amgen Inc.: Employment, Equity Ownership. Woodard:Amgen Inc.: Employment, Equity Ownership.

Abstract 267 Introduction Heparin-induced thrombocytopenia (HIT) is a prothrombotic disorder mediated by platelet, monocyte, and endothelial cell-activating antibodies (Abs) against ultralarge complexes of platelet factor 4 (PF4) and heparin. Laboratory testing plays a key role in the diagnosis of HIT, but is associated with important shortcomings. Immunoassays such as the PF4/heparin ELISA frequently yield false-positive results due to their inability to discriminate cellular activating-Abs from their non-pathogenic counterparts. Functional assays such as the 14C-serotonin release assay (SRA) are more specific, but are unfeasible for most clinical laboratories due to the requirement for radioisotope and fresh platelets from reactive donors. KKO is a monoclonal Ab that causes a HIT-like thrombocytopenic disorder in a mouse model. Binding of KKO to immobilized PF4/heparin is inhibited by human HIT plasma, but not by plasma from patients with non-pathogenic anti-PF4/heparin Abs.1 We exploited this property of KKO to develop a KKO-inhibition (KKO-I) ELISA to detect platelet-activating Abs. We recently described a system to measure cell activation by HIT Abs: DT40 (chicken B lymphocyte) cells transfected with human FcgRIIa coupled to a luciferase reporter.2 We hypothesized that this system (DT40-luc) could be used to identify cell-activating anti-PF4/heparin Abs without need for donor platelets or radioactivity. Here we describe the KKO-I and DT40-luc assays and compare their performance to two commercially available immunoassays and the SRA in samples from 58 patients with suspected HIT and circulating anti-PF4/heparin Abs. Methods Patient samples consecutively referred to a clinical coagulation laboratory for HIT laboratory testing that tested positive by polyspecific anti-PF4/heparin ELISA were included. In addition to the polyspecific ELISA, citrated plasma samples from all patients were tested by an IgG-specific PF4/heparin ELISA, an in-house SRA, and the investigational KKO-I and DT40-luc assays. A 4Ts score to estimate the clinical likelihood of HIT was determined for each subject. The investigator performing 4Ts scoring was blinded to the results of HIT laboratory assays. Investigators performing the KKO-I and DT40-luc assays were blinded to the 4Ts score and the results of the SRA and anti-PF4/heparin ELISA. The KKO-I and DT40-luc assays were performed as previously described.1,2 HIT was defined as the combination of an intermediate or high probability 4Ts score (≥4) and a positive SRA. The performance of each assay with respect to this reference standard was evaluated by receiver-operating characteristic (ROC) analysis. Areas under the ROC curves (AUCs) were calculated and compared using the Delong method for correlated samples. Results Fifty-eight subjects were enrolled, 21 of whom met prespecified criteria for HIT. There were no significant differences in demographic characteristics between the 21 HIT-positive and 37 HIT-negative subjects. The ability of the polyspecific ELISA, IgG-specific ELISA, KKO-I, and DT40-luc assay to discriminate HIT-positive from HIT-negative subjects is shown in Figure 1. HIT-positive plasma showed significantly greater mean inhibition of KKO binding than HIT-negative plasma (70.1%, 95% CI 64.8–75.4 vs. 40.4%, 33.5–47.4, p ROC curves for each assay are shown in Figure 2. The AUC for KKO-I (0.92, 0.85–1.00) was significantly greater than the AUC for the polyspecific (0.82, 0.70–0.95) and IgG-specific (0.76, 0.62–0.90) ELISAs (p Conclusion KKO-I and DT40-luc showed better discrimination than commercially available ELISAs in a small cohort of patients with suspected HIT and anti-PF4/heparin Abs. These assays are simple to perform, do not require donor platelets or radioactivity, and hold promise for improving the specificity and feasibility of HIT laboratory testing. Further evaluation in a larger cohort of patients is required. Disclosures: Cuker: Baxter: Consultancy, Research Funding; Bayer: Consultancy; Canyon: Consultancy; Daiichi Sankyo: Membership on an entity's Board of Directors or advisory committees; Stago: Research Funding. Arepally:Teva Pharmaceuticals: Research Funding. Cines:Amgen: Consultancy; GSK: Consultancy; Eisai: Consultancy; T2 Biosystems: Research Funding.

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 1118 There are no currently available tests to rapidly assess and reliably identify both impaired hemostasis and hypercoagulable states, in part because of difficulties in measuring integrated reactions in whole blood using a single sensitive and clinically useful platform. Methods like T2 magnetic resonance (T2MR) can provide rich information from complex samples, such as changes in the blood during hemostasis, by measuring signals emanating from the hydrogen nuclei within the sample, primarily in water. We used a 14”x6”x7” portable instrument to measure changes in T2MR that provide a continuous report on the dynamically changing microscopic environment of water during coagulation of whole blood (WB), platelet-poor or platelet-rich plasma (PRP). In these initial foundational studies, we measured T2MR continuously over 20 mins using 34 μL blood samples from consented normal adult donors. We tested clotting of WB initiated by the addition of thrombin or kaolin + calcium. Platelet activation was achieved in WB by addition of ADP or arachidonic acid in the presence of reptilase and factor XIIIa with and without addition of the platelet inhibitors aspirin or 2-methylthioadenosine 5'-monophosphate (2-MeSAMP) and results were confirmed by standard platelet aggregometry. At normal platelet counts from 1.5–3×105/μL and normal hematocrit (HCT) from 38%-48%, T2MR gave two curves corresponding to: (1) water trapped within a retracted clot and (2) water in the surrounding liquid, i.e. serum (Fig. 1a). Platelet counts Our proof-of-principle studies show that T2MR technology can be applied to measurement of blood clotting across a range of hemostatic conditions. This single technology may be applicable to the study, diagnosis, and management of a spectrum of disorders that range from impaired hemostasis to hypercoagulable states. These T2MR studies represent the first application of this technology to hemostasis and thrombosis. Additional studies will be needed to develop a more complete understanding of the biochemical events measured by T2MR and to more fully explore its clinical utility. Figure 1. Examples of T2MR 3D surfaces for 34 μL of citrated whole blood mixed activated by adding a dried formulation of CaCl2 (11 mM) and kaolin ( Disclosures: Cines: T2 Biosystems: Research Funding. Lebedeva:T2 Biosystems: Research Funding. Massefski:T2 Biosystems: Employment. Papkov:T2 Biosystems: Employment. Thayer:T2 Biosystems: Employment. Lowery:T2 Biosystems: Employment.