Showing papers in "Seminars in Thrombosis and Hemostasis in 2004"

Platelet dysfunction in renal failure.

Abstract: Patients with end-stage renal disease suffer from complex hemostatic disorders. Uremic patients show a bleeding diathesis that is mainly due to abnormalities of primary hemostasis; in particular, platelet dysfunction and impaired platelet-vessel wall interaction. However, despite decreased platelet function, these patients have a high prevalence of cardiovascular and thrombotic complications. Platelet dysfunction in uremic patients is partially due to uremic toxins present in circulating blood. Dialysis improves platelet abnormalities and reduces, but does not eliminate, the risk of hemorrhage. Hemodialysis can even contribute to the bleeding through the continuous platelet activation induced by the interaction between blood and artificial surfaces. Thrombocytopenia, glomerular thrombosis, and thrombi in small arteries and glomerular capillaries are common pathological features in many renal diseases. Platelets are also involved directly in the pathogenesis of glomerular diseases through a variety of mechanisms, including release of active molecules, by enhancing immune complex deposition, and by altering glomerular permeability.

Platelets and angiogenesis in malignancy.

Abstract: There is increasing evidence that platelets play an important role in the process of tumor angiogenesis. Thrombocytosis is a frequent finding in cancer patients (10-57%). Although the mechanisms underlying thrombocytosis are not yet fully elucidated, tumor-derived factors with thrombopoietin-like activity and growth factors, platelet-derived microparticles, and factors secreted from bone marrow endothelial cells, as well as growth factors released by megakaryocytes (acting via an autocrine loop), are postulated to influence this process. The progression of cancer is associated with hypercoagulability, which results from direct influences of tumor cells and diverse indirect mechanisms. Activated platelets serve as procoagulant surfaces amplifying the coagulation reactions. It is well known that hemostatic proteins are involved in different steps of the angiogenic process. Furthermore, platelets adhering to endothelium facilitate adhesion of mononuclear cells (which exert various proangiogenic activities) to endothelial cells and their transmigration to the extravascular space. It was also documented that platelets induce angiogenesis in vivo. Platelets are a rich source of proangiogenic factors. They also store and release angiogenesis inhibitors. In addition, platelets express surface growth factor receptors, which may regulate the process of angiogenesis. Platelets also contribute directly to the process of basement membrane and extracellular matrix proteolysis by releasing proteinases, or indirectly via inducing endothelial cells and tumor cells to release proteolytic enzymes, as well as through the proteolytic activities of platelet-derived growth factors. The multidirectional activities of platelets in the process of new blood vessel formation during tumor development and metastasis formation may create the possibility of introducing antiplatelet agents for antiangiogenic therapy in cancer patients. Thus far experimental studies employing inhibitors of glycoprotein IIb-IIIa have yielded promising results.

Pathobiology of atherosclerosis--a brief review.

Abstract: Considerable progress has been made recently in understanding the pathobiology of atherosclerosis To a significant degree it is an inflammatory disease of the vessel wall Progression of atherosclerosis or its stabilization reflects the tension between cytokines and effectors that play both an inhibiting and a facilitating role in the progression of atherosclerosis, including platelet-derived growth factor (PDGF), interleukin-1, tumor necrosis factor (TNF) -alpha, and MCP-1 The response to injury model remains central to our understanding of atherogenesis Numerous factors may initiate endothelial injury, including mechanical factors (hypertension and high shear stress in the artery), homocysteine, oxidized low-density lipoprotein (LDL), possibly infectious agents such as Chlamydia, viruses, and toxins such as nicotine These factors lead to endothelial cells' increasing expression of receptors for LDL and increased adherence of monocytes and macrophages and T cells Progression of atherosclerosis can lead to the development of a plaque that is vulnerable to rupture and that would then produce an acute coronary syndrome In addition to standard biomarkers and angiographic approaches for detecting plaque rupture, novel diagnostic approaches are under development, including near infrared spectroscopy, catheter-based thermography, and optical coherence tomography Our better understanding of the atherosclerotic plaque provides multiple opportunities for interdicting arterial injury, and the response to it

Platelet secretory mechanisms.

Abstract: Platelet granule secretion or exocytosis is required for normal platelet function and plays an important role in the pathogenesis of cardiovascular diseases. Platelets secrete molecules that amplify thrombosis, induce vascular remodeling, recruit and activate cells. The platelet secretory process begins in megakaryocytes where molecules are targeted to developing granules through specific vesicle trafficking and transporter mechanisms. Secretory granules may continue to mature in the circulation after the platelet has been released from the megakaryocyte. The platelet secretory process culminates when ligands interact with specific platelet receptors to trigger exocytosis. A convergence of new insights from several different organisms has begun to illuminate the molecular mechanisms responsible for the platelet secretory process, from granule development through membrane fusion and exocytosis.

Molecular defects that affect platelet dense granules.

Abstract: Platelet dense granules form using mechanisms shared by melanosomes in melanocytes and by subsets of lysosomes in more generalized cells. Consequently, disorders of platelet dense granules can reveal how organelles form and move within cells. Models for the study of new vesicle formation include isolated delta-storage pool deficiency, combined alphadelta-storage pool deficiency, Hermansky-Pudlak syndrome (HPS), Chediak-Higashi syndrome, Griscelli syndrome, thrombocytopenia absent radii syndrome, and Wiskott-Aldrich syndrome. The molecular bases of dense granule deficiency are known for the seven subtypes of HPS, as well as for Chediak-Higashi syndrome, Griscelli syndrome, and Wiskott-Aldrich syndrome. The gene products involved in these disorders help elucidate the generalized process of the formation of vesicles from extant membranes such as the Golgi.

Platelet factor 4: an inhibitor of angiogenesis.

Abstract: Platelet factor 4 (PF4) is an antiangiogenic ELR-negative chemokine. PF4 inhibits endothelial cell proliferation and migration and angiogenesis in vitro and in vivo. Three different mechanisms have been proposed to explain PF4's antiangiogenic effects. First, PF4 may bind proteoglycans and interfere with the proteoglycan-bystander effect on growth factor activity. Second, PF4 is able to interact directly with angiogenesis growth factors such as fibroblast growth factors or vascular endothelial growth factors and inhibits their interaction with cell surface receptors. Third, PF4 may activate cell surface receptors on endothelial cells and induce inhibitory signals. Recently, one such receptor, CXCR3-B, was identified. In cardiovascular disease, PF4 may possibly intervene in collateral vessel formation, plaque neovascularization, heparin-induced thrombocytopenia and stent endothelialization. Several PF4 fragments such as PF4-CTF and modified molecules have been made that exhibit antiangiogenesis properties and may serve as leads for further therapeutic development.

Change in protein phenotype without a nucleus: translational control in platelets.

Abstract: For most cells the nucleus takes center stage. Not only is it the largest organelle in eukaryotic cells, it carries most of the genome and transcription of DNA to RNA largely takes place in the nucleus. Because transcription is a major step in gene regulation, the absence of a nucleus is limiting from a biosynthetic standpoint. Consequently, the anucleate status of platelets has stereotyped it as a cell without synthetic potential. It is now clear, however, that this viewpoint is far too simplistic. In response to physiologic stimuli, platelets synthesize biologically relevant proteins that are regulated via gene expression programs at the translational level. This process does not require a nucleus; instead, it uses mRNAs and other translational factors that appear to be retained in specialized fashion as megakaryocytes generate platelets during thrombopoiesis. We highlight the molecular machinery and pathways used by platelets to translate mRNA into protein and offer insight into how these synthesized products may regulate thrombotic and inflammatory events.

Application of flow cytometry to platelet disorders.

Abstract: Flow cytometry is a powerful and versatile tool that can be used to yield definitive information regarding the phenotypic status of platelets. The method provides a quantitative assessment of the physical and antigenic properties of platelets (e.g., surface expression of receptors, bound ligands, components of granules, or interactions of platelets with other platelets, other blood cells, or components of the plasma coagulation system), thereby facilitating the diagnosis of inherited or acquired platelet disorders (e.g., Bernard-Soulier syndrome, Glanzmann thrombasthenia, storage pool disease), the pathological activation of platelets (e.g., in the setting of acute coronary syndromes, cerebrovascular ischemia, peripheral vascular disease, cardiopulmonary bypass), and changes in the ability of platelets to activate via specific stimuli (e.g., efficacy of antiplatelet therapies). Accordingly, this review summarizes the key technical and methodologic components of flow cytometric analysis of platelets, as well as specific examples of its application to diagnosis and patient care.

Role of thrombin in angiogenesis and tumor progression.

Abstract: Clinical, laboratory, histopathological, and pharmacological evidence support the notion that the coagulation system, which is activated in most cancer patients, plays an important role in tumor biology. Our laboratory has provided evidence that thrombin activates angiogenesis, a process which is essential in tumor growth and metastasis. This event is independent of fibrin formation. At the cellular level many actions of thrombin can contribute to activation of angiogenesis: (1). Thrombin decreases the ability of endothelial cells to attach to basement membrane proteins. (2). Thrombin greatly potentiates vascular endothelial growth factor- (VEGF-) induced endothelial cell proliferation. This potentiation is accompanied by up-regulation of the expression of VEGF receptors (kinase insert domain-containing receptor [KDR] and fms-like tyrosine kinase [Flt-1]). (3). Thrombin increases the mRNA and protein levels of alpha (v)beta (3) integrin and serves as a ligand to this receptor. Furthermore, thrombin increases the secretion of VEGF and enhances the expression and protein synthesis of matrix metalloprotease-9 and alpha (v)beta (3) integrin in human prostate cancer PC-3 cells. These results could explain the angiogenic and tumor-promoting effect of thrombin and provide the basis for development of thrombin receptor mimetics or antagonists for therapeutic application.

Tissue factor and fibrin in tumor angiogenesis

Abstract: The hypercoagulability exhibited by most cancer patients leads to serious complications such as venous thromboembolism and contributes to the pathogenesis of tumor growth and metastasis by promoting angiogenesis. The key player in this vicious cycle is tissue factor (TF), the initiator of blood coagulation. Although TF normally safeguards vascular integrity by inducing hemostasis upon injury, abnormal expression of TF in different tumors and related vascular endothelial cells contributes to unnecessary clot formation in cancer patients. Clotting-dependent induction of tumor angiogenesis is primarily mediated by TF-induced generation of thrombin and subsequent deposition of cross-linked fibrin. A cross-linked fibrin network provides a provisional proangiogenic matrix that facilitates blood vessel infiltration. Clotting-independent mechanisms of TF-induced tumor angiogenesis have also been described, mediated primarily by the cytoplasmic tail of the TF receptor. TF activation could contribute to the venous thromboembolism that has been reported as a complication of the use of novel antiangiogenic agents in combination with chemotherapy. Anticoagulants, such as low-molecular-weight heparin, may act to prevent these complications both by interfering with TF-mediated activation of clotting and by directly down-regulating angiogenesis. Thus, TF may prove to be a novel target for cancer therapy.

An overview of the heparin-induced thrombocytopenia syndrome.

Abstract: Heparin-induced thrombocytopenia (HIT) is caused by heparin-dependent, platelet-activating IgG antibodies that increase thrombin generation in vivo, producing a prothrombotic phenotype. In addition to platelet activation, there is in vitro evidence that activation of endothelium and monocytes occurs, perhaps directly by HIT antibodies, but more likely through activated platelet (or microparticle)-endothelial-leukocyte interactions. Patients with cardiac disease receiving heparin present important diagnostic and therapeutic issues when unexpected thrombocytopenia arises. Concomitant vascular disease burden and intravascular catheter use further increase risk of HIT-associated arterial thrombosis in this patient population. Whether arterial thrombosis simply reflects the "hypercoagulability state" of HIT interacting with diseased or injured arteries, or whether arterial "white clots" reflect additional prothrombotic effects of HIT via endothelial and monocyte activation, remains uncertain. Patients with HIT can also develop deep-vein thrombosis, which can progress to limb loss if coumarin (warfarin) leads to severe protein C depletion (coumarin-induced venous limb gangrene). Therapy for patients strongly suspected to have HIT should focus on inhibiting thrombin (or its generation) pharmacologically. Two direct thrombin inhibitors (lepirudin, argatroban) are approved for treating HIT. When using these agents, coumarin anticoagulation should be delayed pending substantial resolution of thrombocytopenia, before cautiously introducing overlapping coumarin therapy.

Clinical gene transfer studies for hemophilia A.

Abstract: The recent advances in gene transfer technology have expedited the development of gene therapy for the treatment of hemophilia A. Three different U.S. Food and Drug Administration-approved phase I clinical trials had been initiated using different gene therapy approaches each with their own advantages and limitations. In the first gene therapy trial for hemophilia A, a non-viral approach was being explored for patients with severe hemophilia A using ex vivo transfected dermal fibroblast expressing B-domain-deleted factor VIII ( BDD-FVIII). There were no serious adverse events and some patients appeared to have experienced fewer bleeding episodes with very low levels of FVIII near baseline. In the second trial, onco-retroviral vectors expressing BDD-FVIII were injected by peripheral intravenous infusion in adult patients suffering from severe hemophilia A. The procedure was safe and in some patients FVIII-transduced cells were detectable in the peripheral blood for more than a year. Although no sustained FVIII expression was detectable, occasional modest changes in FVIII levels were apparent, and in some cases a reduced bleeding frequency occurred compared with historical rates. In another trial, one patient suffering from severe hemophilia A has been treated with a high-capacity (or gutless) adenoviral vector expressing full-length FVIII, which appeared to have resulted in 1% of normal FVIII levels for several months. However, a transient inflammatory response with hematologic and liver abnormalities was observed. In conclusion, although modest improvements in clinical end points have been detected in some patients in these early phase I trials, further improvements in gene delivery technologies are warranted to bring hemophilia A gene therapy one step closer to reality.

Platelet coagulation-protein interactions.

Abstract: The biochemical mechanisms by which activated platelets participate in exposing receptors for the assembly of enzyme-cofactor-substrate complexes at all stages of the blood coagulation cascade are reviewed. Information derived from studies conducted during the last 30 years supports the concept that the initiation of blood coagulation is triggered by exposure of tissue factor at injury sites, leading to the generation of minute quantities of thrombin (limited by tissue factor pathway inhibitor), sufficient to activate platelets, factors XI, VIII, and V, and trigger the consolidation pathway (i.e., the sequential activation of factors XI, IX, X, and prothrombin on the activated platelet surface), leading to the generation of sufficient thrombin to convert fibrinogen to fibrin and effect hemostasis. Platelets localize coagulation to the hemostatic thrombus and protect coagulation enzymes from inhibition by both plasma and platelet inhibitors (e.g., protease nexin 2), thus preventing disseminated intravascular coagulation.

Inherited defects in platelet signaling mechanisms.

Abstract: In the majority of patients with an inherited abnormality in platelet function and a bleeding diathesis, the underlying platelet molecular mechanisms are unknown. The usually considered entities, such as thrombasthenia, the Bernard-Soulier syndrome, and storage pool deficiency, occur in a small proportion of patients. A substantial number of patients present with decreased aggregation and secretion of dense granule contents upon activation, and are lumped in the category of primary secretion defects or platelet activation defects. Evidence is now available that defects in platelet signaling mechanisms may be the basis for the platelet dysfunction in some of these patients. This evidence is presented here. If the key components in signal transduction are the surface receptors, the G-proteins, and the effectors, evidence now exists for specific human platelet abnormalities at each of these levels. There is a pressing need for a concerted effort to delineate the molecular mechanisms in the large group of patients with impaired platelet function who represent an untapped reservoir of new information into normal platelet function.

Clinical gene transfer studies for hemophilia B.

Abstract: Hemophilia B, a deficiency of functional factor IX (FIX), has been extensively explored as a model for gene transfer. Two U.S. Food and Drug Administration-approved clinical studies for hemophilia B have been undertaken, both using adeno-associated viral vectors (AAV). AAV vectors have tropism for liver, muscle, central nervous system, and the respiratory tract; both skeletal muscle and liver have been used as target tissues in the hemophilia B studies. In both studies, proof of principle was first established in the hemophilia B dog model, with long-term expression of canine FIX at therapeutic levels achieved before clinical studies were initiated. In the AAV-FIX muscle trial, vector was introduced into skeletal muscle of the upper and lower extremities of eight human patients by direct intramuscular injection. Muscle biopsies taken 2 to 10 months postinjection demonstrated gene transfer and expression (by Southern blot and immunofluorescence, respectively) in all patients, but circulating FIX levels were generally not >1%, and escalation of dose to levels that proved therapeutic in animals was thwarted by feasibility issues regarding the number of injections required. Nevertheless, the study demonstrated that parenteral injection of AAV-FIX was safe at the doses tested, and could result in long-term expression of the transgene. Moreover, the general characteristics of transduction of human muscle were similar to those observed in other animal models. The safety and efficacy data established in the first trial formed the basis for a second trial in which AAV-FIX is administered systemically to target the liver. The liver study is currently ongoing, with six patients enrolled to date.

Generic low-molecular-weight heparins: some practical considerations.

Abstract: It is now widely accepted that various low-molecular-weight heparins (LMWHs) exhibit specific molecular and structural attributes that are determined by the type of manufacturing process used. For example, enoxaparin, which is prepared by benzylation followed by alkaline hydrolysis of unfractionated heparin (UFH), exhibits a double bond at the nonreducing end and the presence of a unique bicyclic structure namely 1,6 anhydromanno glucose or mannose, or both, at the reducing end. Similarly, the other LMWHs, such as dalteparin, nadroparin, tinzaparin, and parnaparin, exhibit specific structural characteristics that may contribute to their own unique biochemical and pharmacological profiles. These unique features may not exhibit any major influence on the routinely determined anti-Xa and anti-IIa activities. However, these may have an impact on the pharmacokinetics and other biological actions such as the interactions with growth factors, blood components, and vascular cells. This is the reason for the initial caution for the noninterchangeability of the anti-Xa adjusted dosing of the different LMWHs. Although the nonanticoagulant biological effects of these drugs are poorly understood at this time, they are now recognized as contributing significantly to the overall therapeutic effects of these drugs. Because some of these drugs have proved to be effective in the management of cancer-associated thrombosis and exhibit improvements in mortality outcome, these LMWHs may also produce several other effects by modulating inflammatory processes, apoptosis, and other regulatory functions related to cellular functions at different levels. Thus, the interactions of these LMWHs with antithrombin and heparin cofactor II are not the only determinants of their biological actions. Release of tissue factor pathway inhibitor (TFPI), regulation of cytokines, nitric oxide, and eicosanoids contribute to their individuality. Such properties are not only dependent on the oligosaccharide sequence and consensus sites but also depend mainly on microchemical and structural attributes in these drugs. European Pharmacopoeia (EP) and the World Health Organization (WHO) have developed guidelines to characterize these agents in terms of their molecular and biological profile. Regulatory agencies such as the U.S. Food and Drug Administration (FDA) and European Medicines Agency (EMEA) consider each of these drugs as distinct pharmacological agents. This has prompted the requirement for product-specific clinical data for the approval of their use in various clinical indications. There is a clear concern regarding the development of potential generic versions of branded products and the submissions by generic manufacturers for the regulatory approval of generic interchangeability that refers to the substitution of an apparent chemically identical and bioequivalent versions of the branded LMWHs. Currently, there are no regulatory guidelines or consensus opinions on the acceptance of generic versions of the branded products. Because the LMWHs represent not only a biological entity but also product-specific molecular and structural attributes, the acceptance of a generic version must be based on clearly defined guidelines stipulating minimal molecular and structural, biological, and clinical validation requirements. It is therefore to be stressed that each of the LMWHs is a distinct drug entity that characteristically exhibits a product-based therapeutic spectrum in different thrombotic and nonthrombotic disorders. Thus, until the establishment of valid regulatory guidelines for the generic interchangeability of the commercially available LMWHs is completed, generic substitutes are not recommended.

Contribution of the hemostatic system to angiogenesis in cancer.

Abstract: It is now recognized that the hemostatic system plays an important role in cancer growth and dissemination, processes known to be vitally dependent on new tumor blood vessel formation (angiogenesis). There is also an increasing body of evidence supporting the link between the various components of the coagulation/fibrinolysis systems and angiogenic activity in cancer patients. Tissue factor (TF), thrombin, fibrinogen, fibrin, and plasminogen activation system, as well as platelets, all are able to promote angiogenesis. On the other hand, coagulation inhibitors, as well as cryptic (proteolytically released) domains of hemostatic proteins, are also known to act as angiogenesis inhibitors. Indeed, modulation (stimulation or inhibition) of angiogenesis may result from either classical functions of various molecular components of the hemostatic cascade, their less studied "alternative" activities, or both. Although much remains to be understood about this complex circuitry these considerations support the judicious use of anticoagulants in patients with malignancy as well as encourage the search for novel antiangiogenic activities that may reside within molecular and cellular components of the hemostatic system.

Platelet-collagen responses: molecular basis and therapeutic promise.

Abstract: Exposure of circulating blood to collagen after vessel injury has been recognized as a primary event in arterial thrombosis for many years but an understanding of the molecular basis of this response has only recently materialized. Platelet collagen interactions are initiated indirectly by interaction of platelet glycoprotein Ib (GPIb) with collagen-bound von Willebrand Factor (vWF). Slowed, rolling platelets firmly adhere following the generation of platelet activating signals in a process initiated by the platelet collagen receptor glycoprotein VI (GPVI). The contribution of the first platelet collagen receptor identified, integrin alpha (2)beta (1), remains controversial. alpha (2)beta (1) is not required for platelet responses to collagen under many experimental conditions but GPVI and other intracellular signals may activate the integrin, which is likely to play a later, secondary role. In vivo studies of arterial thrombosis using genetically modified mice suggest that blockade of platelet-collagen interactions may be a highly effective means of inhibiting arterial thrombosis. Recent studies have generated a more complete model of the molecular basis of platelet-collagen responses and provided both the means and incentive to create novel therapeutic agents aimed at blocking this process to treat human atherothrombotic diseases such as myocardial infarction and stroke.

Platelet receptors for adenine nucleotides and thromboxane A2.

Abstract: Adenosine diphosphate (ADP) and thromboxane A (2) (TXA (2)) are important physiological activators of platelets and exert their effects by acting on cell surface receptors Platelet nucleotide receptors can be distinguished as three separate subtypes of the P2 receptor family The P2X (1) receptor is a ligand-gated adenosine triphosphate (ATP) receptor that was originally mistaken for an ADP receptor This calcium-influx-causing receptor mediates platelet shape change and plays an important role in thrombus formation in small arterioles The P2Y (1) receptor, through activation of G (q) and phospholipase C, is required for ADP-induced platelet shape change, fibrinogen receptor activation, and TXA (2) generation The G (i)-coupled P2Y (12) receptor plays an important role in platelet aggregation, potentiation of dense granule release, and TXA (2) generation Both the P2Y receptors are crucial for in vivo thrombus formation TXA (2) stimulates two subtypes of G protein-coupled TP receptor, TPalpha and TPbeta, but its effects in platelets are mediated predominantly through the alpha isoform Although interference with the activation of G protein-coupled ADP or TP receptors results in increased bleeding times and protection from thromboembolism, TP receptor antagonists did not translate into effective antiplatelet drugs Blockade of ADP receptor is a mode of newer classes of antithrombotic drugs in the coming era This review focuses on the contribution of different nucleotide receptors and TP receptors to platelet function and their potential as antithrombotic agents

Endothelial cell development, vasculogenesis, angiogenesis, and tumor neovascularization: an update.

Abstract: In the embryo, blood vessel formation de novo (vasculogenesis) and from existing vessels (angiogenesis) results in blood vessels lined by endothelial cells (ECs). The relationship between ECs and blood cells suggested by their physical closeness was recently confirmed with the demonstration of progenitors that give rise to both cell types. In tumors, new blood vessel formation has been thought to occur primarily via angiogenesis. Recent evidence, however, suggests that postnatal vasculogenesis also contributes to tumor neovascularization. In this article, we provide an update on EC development, including early lineage specification, morphogenesis or differentiation to form functional blood vessels, and regulation of EC survival and senescence. Furthermore, we review the latest findings on tumor neovascularization and therapeutic potentials of molecules critical to this process.

Oncogenes as regulators of tissue factor expression in cancer: implications for tumor angiogenesis and anti-cancer therapy.

Abstract: Up-regulation of tissue factor (TF) is often observed in cancer. TF is a cell-associated receptor for coagulation factor VII/VIIa, an interaction known to activate the coagulation cascade. At the same time, TF is also known as a mediator of intracellular signaling events that can alter gene expression patterns and cell behavior. Both aspects of TF activity are of possible relevance to tumor growth, metastasis, and angiogenesis, including up-regulation of vascular endothelial growth factor (VEGF). TF up-regulation is often observed on the surfaces of tumor-associated endothelial cells, inflammatory cells, and particularly on cancer cells themselves. In the last case, high TF levels may be associated with poor prognosis and parallel clinical (and genetic) tumor progression. We have proposed elsewhere that TF may be a target of oncogenic events in cancer. Here we discuss our observations suggesting that oncogene-targeting agents may down-regulate TF expression. Such is the effect of treatment with the neutralizing monoclonal antibody (C225) raised against the epidermal growth factor receptor (EGFR) in EGFR-dependent squamous cell carcinoma cells (A431). This two- to threefold TF down-regulation by C225 treatment is paralleled by a decrease in expression of VEGF. It is conceivable that TF participates in signals that regulate VEGF and angiogenesis triggered by activated oncogenic pathways. Therefore, direct targeting of TF in cancer should be considered in combination with other treatment modalities such as oncogene-directed therapies, antiangiogenic agents (e.g., VEGF antagonists), and anti-cancer chemotherapy.

Monitoring the new antithrombotic drugs.

Abstract: Today there is a diverse group of anticoagulant and antithrombotic drugs available that includes warfarin derivatives, heparin, low-molecular-weight heparins, thrombin inhibitors, factor Xa inhibitors, and various antiplatelet agents. Many of these new drugs do not alter measurable blood coagulation parameters, yet they are effective antithrombotic agents through their actions on vascular endothelial cells and proteins. Thus, these new agents do not affect the traditional clot-based prothrombin time/International Normalized Ratio (PT/INR) and activated partial thromboplastin time (aPTT) tests, and monitoring and standardization require the development of new methods. In addition to clot-based assays, chromogenic assays, enzyme-linked immunosorbent assay (ELISA), high-performance liquid chromatography (HPLC), flow cytometry, and other techniques have been used to monitor these new drugs. On the other hand, some of the new antithrombotic drugs do affect the PT, aPTT, and activated clotting time (ACT); however, they behave differently from the warfarin derivatives and heparin. The traditionally used relationship of target time to clot values and INR to clinical effect cannot necessarily be transferred to the new drugs. Unfortunately, monitoring is not as simple as it was for warfarin and heparin. Although the new antithrombotic drugs have been approved for clinical use, assay systems for monitoring most of them are still in development or have not been clinically validated. This applies to each of the clinical settings targeted for prophylaxis, treatment, or interventional procedures (i.e., high- and low-dosing regimens typically require different monitoring methods). In addition to basic monitoring, other issues such as sensitivity of the drug to different laboratory monitoring reagents and instrumentation, drug combination monitoring, and patient-related factors that contribute to the variability of the results still need to be addressed.

Immune implications of gene therapy for hemophilia.

Abstract: Similar to any novel treatment strategy for hemophilia, gene therapy faces the question of the risk of formation of inhibitory antibodies to the therapeutic factor VIII or factor IX protein. Activation of CD4 (+) or CD8 (+) T cells could lead to antibody formation or cytotoxic T lymphocyte responses to transgene-expressing cells. Preclinical studies in animal models of hemophilia A and B with different mutations in the dysfunctional gene shed light on the risk for such immune responses and point toward strategies to avoid immune activation or even promote tolerance induction. The impacts of variables such as choice and design of gene transfer vector, underlying gene mutation, route of vector administration, and transient immune suppression are discussed. Maintenance of immunological hyporesponsiveness to the therapeutic gene product is critical for successful gene therapy. Recent studies provide evidence for tolerance induction to coagulation factor antigens by viral hepatic or neonatal in vivo gene transfer, by in utero gene delivery, and by oral or nasal administration of protein or peptides.

Decreased prevalence of heparin-induced thrombocytopenia with low-molecular-weight heparin and related drugs.

Abstract: Heparin-induced thrombocytopenia (HIT) Type II represents a disease spectrum associated with a high risk of thrombosis leading to limb loss and death. The pathophysiology of HIT is based on the development of antibodies to the heparin-platelet factor 4 (PF4) complex. Unfractionated heparin (UFH) is heterogeneous in molecular chain length and degree of sulfation accounting in part, for, the heterogeneity of HIT antibodies. Because of its smaller size, low-molecular-weight heparin (LMWH) does not interact with PF4 and platelets as efficiently as does UFH. This translates into a lower risk of immune sensitization with LMWH than with UFH treatment. LMWH is less likely than UFH to cause antibody generation and thus patients do not develop clinical HIT at the same frequency with LMWH as with UFH treatment. The antibodies generated by LMWH treatment are more often immunoglobulin A (IgA) and IgM as opposed to IgG antibodies, which are associated with symptomatic clinical HIT generated by exposure to UFH. However, platelet activation/aggregation can occur from LMWHs in the presence of most pre-existing HIT antibodies that had been generated from UFH exposure, although the response is less than that caused by UFH plus HIT antibody. With the expanded use of LMWH, the frequency of clinical HIT may naturally decline, given that LMWHs are less likely to generate HIT antibody.

Interactions of platelet factor 4 with the vessel wall.

Abstract: Platelet factor 4 (PF4) is a platelet-specific protein that is stored in platelet alpha granules and released following platelet activation. PF4 was the first chemokine that was isolated, but unlike other chemokines, it may not have a clear role in inflammation. Gathering evidence suggests that unlike other chemokines that bind to specific receptors, PF4's biology depends on its unusually high affinity for heparan sulfates and other negatively charged molecules at concentrations attained in the immediate vicinity of activated platelets. There has been one report that PF4 binds to CXCR3B, a chemokine receptor isoform that may be present in some vascular beds, but the biological relevance of this single observation is not clear. We propose that the main biological role of PF4 and the basis for its presence in the alpha granules of all known mammalian platelets is to neutralize surface heparan sulfate side-chains of glycosaminoglycans and to optimize thrombus development at sites of vascular injury. In addition, the binding of PF4 to surface glycosaminoglycans may also underlie its angiostatic and proatherogenic properties. Additionally, PF4 binds to several other proteins that are central to thrombosis, angiogenesis, and atherogenesis. These interactions may also contribute to its biological and pathobiological effects. Certainly, future studies using in vivo models to test biological relevance of each of these proposed mechanisms by which PF4 interacts with the vasculature are needed, as are studies to define the importance of PF4 binding to CXCR3B.

A critical evaluation of assays for detecting antibodies to the heparin-PF4 complex.

Abstract: Heparin-induced thrombocytopenia (HIT) is a potentially catastrophic complication of heparin therapy The syndrome is the result of the production of an antibody to the complex that forms between heparin and platelet factor 4 (H-PF4) released from activated platelets IgG antibodies bind to platelet Fc receptors and cause platelet activation, resulting in thrombocytopenia and greatly increased risk of thrombosis Tests for the H-PF4 antibody can be classified into functional assays (which rely on the demonstration of platelet activation) and immunoassays (which detect the presence of an antibody without regard for its functional ability) The functional assays have a greater specificity for clinical HIT, but require normal donor platelets and are relatively un-standardized The immunoassays have the advantage of better standardization and do not require normal platelets, but may give positive results in the absence of clinical HIT The choice of test is usually dictated by what is possible for a given laboratory in terms of instrumentation, expertise, and interest For most institutions this will be a commercially available enzyme-linked immunosorbent assay Although HIT is a true clinicopathological syndrome, there are several reasons why its diagnosis still rests primarily on clinical grounds First, laboratory tests may not be available locally Second, they may not be available in a sufficiently timely manner Finally, available tests are not completely sensitive or specific for the condition There is, therefore, a continuing need to develop more rapid testing strategies with greater specificity for clinical HIT

Eicosanoid Regulation of Angiogenesis in Tumors

Abstract: Tumor angiogenesis, the formation of new capillary blood vessels in tumors from pre-existing vasculature, is required for tumor growth and progression. Eicosanoids, the bioactive lipids derived from arachidonic acid, possess potent and diverse biological activities. In response to stimuli, arachidonic acid is mobilized from phospholipid pools and metabolized by cyclooxygenases (COX), lipoxygenases (LOX), and p450 epoxygenases (EOX) to form a variety of eicosanoids. The involvement of eicosanoids in tumor angiogenesis and progression is implicated by the observations that nonsteroidal anti-inflammation drugs (NSAIDs) reduce tumor growth and angiogenesis. Subsequently, it is found that the levels of COX-2 and/or 12-LOX are frequently increased in various cancers. Further studies using molecular and pharmacological approaches have found that COX-2 and 12-LOX, when overexpressed in carcinoma cells, enhance their angiogenic potential and stimulate tumor growth. In this article, we discuss how COX and LOX in cancer cells modulate tumor angiogenesis and present the possibility of using NSAIDs and LOX inhibitors as antiangiogenesis agents.

Newer insights on the mechanism of heparin-induced thrombocytopenia.

Abstract: Heparin-induced thrombocytopenia (HIT) type II is a complex clinical syndrome. It is an immune reaction to heparin in which the formation of antibodies targeted against the heparin-platelet factor 4 complex results in platelet activation. Platelet activation plays a central role in HIT; however, platelet activation does not occur as an isolated physiologic response. To elucidate further the mechanism of thrombogenesis in HIT, we undertook studies to determine the effect of heparin antibodies on endothelial cells, leukocytes, and the inflammatory state. We summarize our previous and new findings. For endothelial cells: Antiheparin antibodies bind to and directly activate microvascular endothelial cells, whereas binding to and activating macrovascular endothelial cells requires preactivation by platelets or tumor necrosis factor alpha (TNFalpha). Increased circulating levels of hemostatic activation factors as observed with thrombosis, particularly soluble P-selectin, plasminogen activator inhibitor type 1 (PAI-1), tissue factor, and thrombomodulin, were associated with endothelial cell activation and were also found in the blood circulation of patients with HIT. For the inflammatory state: Neutrophils and monocytes (but not lymphocytes) bind to and form complexes with platelets in the presence of HIT antibodies. Activated monocytes bind to endothelial cells and produce a procoagulant state. Patients with HIT have an increased level of cytokines in their blood circulation. For HIT antibodies: Only heparin fractions larger than 5 kd interacted with HIT antibodies, explaining why low-molecular-weight heparin (LMWH) usually does not generate antibodies. HIT antibodies are heterogeneous in structure, affinity, and specificity. These data suggest that, in addition to the platelet component, several other mechanisms are associated with the pathophysiology of HIT. These include an inflammatory state, endothelial cell remodeling, and the known procoagulant state. Differences between patients in the levels of the inflammatory markers may relate to various stages of the inflammatory/procoagulant state that exists in patients with HIT. The variations within the HIT antibodies may influence their ability to activate platelets, endothelial cells, and leukocytes, and thus contribute further to the variations in the pathogenicity of HIT.

Low-molecular-weight heparin in pediatric patients.

Abstract: The incidence of thromboembolic events (TEs) in childhood is greatly underestimated. Two age groups account for ∼70% ofTEs in childhood: infants and teenagers. There are several predisposing risk factors for newborns such as small vessels, high hematocrit, and a unique neonatal hemostatic system. Central venous lines contribute to 80% of deep vein thrombosis in newborns. Other risk factors for all children are shock syndromes, trauma, surgery, heart and kidney disease, and acquired or hereditary thrombophilias. The best prophylaxis is to recognize, avoid, and remove risk factors if possible. This is particularly relevant in childhood, where risk factors can be found in the majority of TEs. The serious sequelae of TEs (mortality, and short- and long-term morbidity) require therapeutic intervention. Unfractionated heparin (UFH) has the following disadvantages: age-dependent unpredictable pharmacokinetics, the need for intravenous access for therapy and monitoring, delays in achieving therapeutic ranges, bleeding risk, the risk of heparin-induced thrombocytopenia, and osteoporosis with long-term use. Oral anticoagulants, in addition to some of these disadvantages, show considerable variation by diet (especially if there is a change from breast to bottle feeding), medication, and intercurrent illness. Review of case reports and cohort studies on 728 children treated with low-molecular-weight heparin (LMWH) indicate the following advantages over UFH: minimal monitoring, ease of administration (subcutaneous), and possibly equivalent efficacy and safety. Dose recommendations for pediatric patients cannot be directly extrapolated from those for adult patients. If dosages are calculated according to body weight, infants < 3 months (or < 5 kg) need ∼50% more LMWH than older children or adults to reach prophylactic or therapeutic anti-factor Xa levels. Further studies are necessary to address the following: the importance of risk factors, the necessity of screening for hereditary thrombophilia, the efficacy and safety of treatment, and side effects and duration of treatment. Thromboembolic events (TEs) are considered to be rare in children. However, recent surveys reveal that TEs in children occur more often than suspected. The incidence is greatly underestimated because TEs are usually overlooked. Retrospective surveys in children treated for acute lymphoblastic leukemia with corticosteroids and asparaginase revealed clinically symptomatic TE in only 2 to 12% of patients. 1 However, in prospective studies with routine imaging, the incidence was more than 20%. 2,3 The objectives of this article are to update the present knowledge on TEs in children, including incidence, predominant age groups, risk factors, diagnosis, and indications for prophylaxis and therapy; and to discuss the use of low-molecular-weight heparin (LMWH) in children.

Platelet transcriptome: the application of microarray analysis to platelets.

Abstract: Human blood platelets are intimately involved in the regulation of thrombosis, inflammation, and wound repair. These cells retain megakaryocyte-derived cytoplasmic mRNA and functionally intact protein translational capabilities, although very little is known about normal or pathological mRNA profiles. Microarray analysis has demonstrated a clear and reproducible molecular signature unique to platelets. There is a relative paucity of expressed transcripts compared with those found in other eukaryotic cells, most likely related to mRNA decay in these anucleate cells. In contrast, a complementary methodology for transcript profiling (serial analysis of gene expression [SAGE]) demonstrates that 89% of tags represent mitochondrial (mt) transcripts (enriched in 16S and 12S ribosomal RNAs), presumably related to persistent mt-transcription in the absence of nuclear-derived transcripts. The abundance of nonmitochondrial SAGE tags parallels relative expression for the most abundant transcripts as determined by microarray analysis, establishing the concordance of both techniques for platelet profiling. These observations establish the validity of transcript analysis as a tool for identifying novel platelet genes that may regulate normal and pathologic platelet (and/or megakaryocyte) functions. The potential application of platelet-specific microarrays in scientific and clinical settings related to platelet production, cardiovascular, and cerebrovascular diseases is reviewed.