James D. McFadyen

Differentiating haemostasis from thrombosis for therapeutic benefit

The central role of platelets in the formation of the primary haemostatic plug as well as in the development of arterial thrombosis is well defined. In general, the molecular events underpinning these processes are broadly similar. Whilst it has long been known that disturbances in blood flow, changes in platelet reactivity and enhanced coagulation reactions facilitate pathological thrombus formation, the precise details underlying these events remain incompletely understood. Intravital microscopy studies have highlighted the dynamic and heterogeneous nature of thrombus development and demonstrated that there are considerable spatiotemporal differences in the activation states of platelets within a forming thrombus. In this review we will consider the factors regulating the activation state of platelets in a developing thrombus and discuss how specific prothrombotic factors may influence this process, leading to excessive thrombus propagation. We will also discuss some potentially novel therapeutic approaches that may reduce excess thrombus development whilst minimising bleeding risk.

The class II PI 3-kinase, PI3KC2α, links platelet internal membrane structure to shear-dependent adhesive function

PI3KC2α is a broadly expressed lipid kinase with critical functions during embryonic development but poorly defined roles in adult physiology. Here we utilize multiple mouse genetic models to uncover a role for PI3KC2α in regulating the internal membrane reserve structure of megakaryocytes (demarcation membrane system) and platelets (open canalicular system) that results in dysregulated platelet adhesion under haemodynamic shear stress. Structural alterations in the platelet internal membrane lead to enhanced membrane tether formation that is associated with accelerated, yet highly unstable, thrombus formation in vitro and in vivo. Notably, agonist-induced 3-phosphorylated phosphoinositide production and cellular activation are normal in PI3KC2α-deficient platelets. These findings demonstrate an important role for PI3KC2α in regulating shear-dependent platelet adhesion via regulation of membrane structure, rather than acute signalling. These studies provide a link between the open canalicular system and platelet adhesive function that has relevance to the primary haemostatic and prothrombotic function of platelets.

Platelets are not just for clots

Although the role of platelets as central mediators of hemostasis and thrombosis has been the primary focus of research into platelet biology for more than a century, over the last decade, nonhemostatic functions of platelets have been increasingly defined. As such, a large body of experimental evidence now exists, which places the platelet as a key player in mediating a diverse range of immune, inflammatory, and malignant disease processes. This review outlines the central mechanisms that underpin the nonhemostatic role of platelets and provides a summary of evidence demonstrating a role for platelets in mediating selected inflammatory, immune, and malignant disease processes.

Current and future antiplatelet therapies: emphasis on preserving haemostasis

Antiplatelet drugs, such as aspirin, P2Y12 antagonists, and glycoprotein (GP) IIb/IIIa inhibitors, have proved to be successful in reducing the morbidity and mortality associated with arterial thrombosis. These agents are, therefore, the cornerstone of therapy for patients with acute coronary syndromes. However, these drugs all carry an inherent risk of bleeding, which is associated with adverse cardiovascular outcomes and mortality. Thus, the potential benefits of more potent, conventional antiplatelet drugs are likely be offset by the increased risk of bleeding. Data from experiments in vivo have highlighted potentially important differences between haemostasis and thrombosis, raising the prospect of developing new antiplatelet drugs that are not associated with bleeding. Indeed, in preclinical studies, several novel antiplatelet therapies that seem to inhibit thrombosis while maintaining haemostasis have been identified. These agents include inhibitors of phosphatidylinositol 3-kinase-β (PI3Kβ), protein disulfide-isomerase, activated GPIIb/IIIa, GPIIb/IIIa outside-in signalling, protease-activated receptors, and platelet GPVI-mediated adhesion pathways. In this Review, we discuss how a therapeutic ceiling has been reached with existing antiplatelet drugs, whereby increased potency is offset by elevated bleeding risk. The latest advances in our understanding of thrombus formation have informed the development of new antiplatelet drugs that are potentially safer than currently available therapies.

Dok-2 Adaptor Protein Regulates the Shear-dependent Adhesive Function of Platelet Integrin αIIbβ3 in Mice

Background: Dok proteins are negative regulators of immunoreceptor signaling and, potentially, integrin adhesion receptors. Results: Deficiency of Dok-2 results in enhanced shear-dependent integrin adhesion in platelets, leading to accelerated platelet thrombus growth. Conclusion: Dok-2 is a shear-specific negative regulator of blood clot formation. Significance: Dok-2 regulates biomechanical platelet adhesion, and targeting this molecule may provide new avenues to regulate thrombosis. The Dok proteins are a family of adaptor molecules that have a well defined role in regulating cellular migration, immune responses, and tumor progression. Previous studies have demonstrated that Doks-1 to 3 are expressed in platelets and that Dok-2 is tyrosine-phosphorylated downstream of integrin αIIbβ3, raising the possibility that it participates in integrin αIIbβ3 outside-in signaling. We demonstrate that Dok-2 in platelets is primarily phosphorylated by Lyn kinase. Moreover, deficiency of Dok-2 leads to dysregulated integrin αIIbβ3-dependent cytosolic calcium flux and phosphatidylinositol(3,4)P2 accumulation. Although agonist-induced integrin αIIbβ3 affinity regulation was unaltered in Dok-2−/− platelets, Dok-2 deficiency was associated with a shear-dependent increase in integrin αIIbβ3 adhesive function, resulting in enhanced platelet-fibrinogen and platelet-platelet adhesive interactions under flow. This increase in adhesion was restricted to discoid platelets and involved the shear-dependent regulation of membrane tethers. Dok-2 deficiency was associated with an increased rate of platelet aggregate formation on thrombogenic surfaces, leading to accelerated thrombus growth in vivo. Overall, this study defines an important role for Dok-2 in regulating biomechanical adhesive function of discoid platelets. Moreover, they define a previously unrecognized prothrombotic mechanism that is not detected by conventional platelet function assays.

Neutrophil macroaggregates promote widespread pulmonary thrombosis after gut ischemia

Neutrophil macroaggregates induce a distinct arterial-venous thrombotic response in the lung after gut ischemia. Rip n’ roll Ischemia in critically ill patients can result in thrombosis of unrelated organs, which is partially due to neutrophil recruitment. Yuan et al. combined intravital microscopy of thrombosis after gut ischemia-reperfusion injury with samples from acute respiratory distress syndrome patients. They observed that rolling neutrophils grab and rip fragments from phosphatidylserine-expressing dying platelets, which leads to macroaggregates. These macroaggregates, in turn, induce thrombosis and were not able to be targeted by conventional therapies such as aspirin. Conversely, targeting the necrotic factor cyclophilin D did have beneficial effects. These studies reveal new thrombotic biology and suggest the development of alternatively targeted therapies to prevent distant organ injury. Gut ischemia is common in critically ill patients, promoting thrombosis and inflammation in distant organs. The mechanisms linking hemodynamic changes in the gut to remote organ thrombosis remain ill-defined. We demonstrate that gut ischemia in the mouse induces a distinct pulmonary thrombotic disorder triggered by neutrophil macroaggregates. These neutrophil aggregates lead to widespread occlusion of pulmonary arteries, veins, and the microvasculature. A similar pulmonary neutrophil-rich thrombotic response occurred in humans with the acute respiratory distress syndrome. Intravital microscopy during gut ischemia-reperfusion injury revealed that rolling neutrophils extract large membrane fragments from remnant dying platelets in multiple organs. These platelet fragments bridge adjacent neutrophils to facilitate macroaggregation. Platelet-specific deletion of cyclophilin D, a mitochondrial regulator of cell necrosis, prevented neutrophil macroaggregation and pulmonary thrombosis. Our studies demonstrate the existence of a distinct pulmonary thrombotic disorder triggered by dying platelets and neutrophil macroaggregates. Therapeutic targeting of platelet death pathways may reduce pulmonary thrombosis in critically ill patients.

Platelet-Derived Microvesicles in Cardiovascular Diseases

Microvesicles (MVs) circulating in the blood are small vesicles (100–1,000 nm in diameter) derived from membrane blebs of cells such as activated platelets, endothelial cells, and leukocytes. A growing body of evidence now supports the concept that platelet-derived microvesicles (PMVs), the most abundant MVs in the circulation, are important regulators of hemostasis, inflammation, and angiogenesis. Compared with healthy individuals, a large increase of circulating PMVs has been observed, particularly in patients with cardiovascular diseases. As observed in MVs from other parent cells, PMVs exert their biological effects in multiple ways, such as triggering various intercellular signaling cascades and by participating in transcellular communication by the transfer of their “cargo” of cytoplasmic components and surface receptors to other cell types. This review describes our current understanding of the potential role of PMVs in mediating hemostasis, inflammation, and angiogenesis and their consequences on the pathogenesis of cardiovascular diseases, such as atherosclerosis, myocardial infarction, and venous thrombosis. Furthermore, new developments of the therapeutic potential of PMVs for the treatment of cardiovascular diseases will be discussed.

Bortezomib-based antibody depletion for refractory autoimmune hematological diseases

Certain patients with antibody-mediated autoimmune disease exhibit poor responses to conventional immunosuppression, including B-cell depletion with rituximab. Proteasome inhibitors such as bortezomib demonstrate pleiotropic immunomodulatory effects, including direct toxicity to antibody-producing cells. Here, we report preliminary evidence for the efficacy of bortezomib as salvage therapy for refractory autoimmune hematological disease. Thirteen treatment episodes in 10 patients with autoimmune hematological phenomena (autoimmune hemolytic anemia [AIHA; n = 8], acquired hemophilia (n = 1), immune thrombocytopenia (n = 1), and thrombotic thrombocytopenic purpura [TTP; n = 3]) and a median of 5 (range, 3-12) prior lines of therapy demonstrated an overall response rate of 77% (10 of 13) including 38% (5 of 13) complete remissions. The majority of clinical improvements were rapid, correlated with biomarkers of autoantibody reduction, and were associated with an acceptable safety profile. Responses appeared durable following treatment of TTP and acquired hemophilia; AIHA responses were more limited with a pattern of relapse following bortezomib cessation. These data provide proof of concept for the utility of proteasome inhibition as antibody depletion therapy in autoimmune disease.

The successful management of dabigatran-associated critical end-organ bleeding with recombinant factor VIIa

Dear Editor, The management of dabigatran-related critical end-organ bleeding is a clinical dilemma due to its increasing clinical use and lack of specific reversal agent. We report a case of dabigatran-related life-threatening intracranial haemorrhage, requiring urgent neurosurgery, which was successfully managed with recombinant factor VIIa (rFVIIa) perioperatively. This is the first case to our knowledge that demonstrates a correction of thrombin generation by rFVIIa in the clinical setting of dabigatran-related major bleeding. A 72-year-old male with atrial fibrillation receiving dabigatran 150 mg twice daily presented with a closed head injury. On presentation, the Glasgow Coma Scale (GCS) was 13, and a computed tomography (CT) of the brain demonstrated an 18-mm subdural haemorrhage with midline shift and contralateral hydrocephalus. Initial coagulation screen revealed a prolonged thrombin clotting time (TCT) of 213 s (reference 0–24 s) and activated partial thromboplastin time (APTT) of 42.2 s (reference 26–35 s). Dabigatran level (Hemoclot assay) on arrival was therapeutic at 146 ng/ml (7 h post-dose) (reference Cpeak 117–275 ng/ml 2 h post-dose) [1]. Renal function was normal. Haemodialysis to reduce the dabigatran level could not be pursued as neurosurgery was required urgently, and 90 μg/kg of rFVIIa, the recommended dosage for haemophilia with inhibitors [2], was administered 30 min preoperatively (10 h postdabigatran ingestion). Both rFVIIa and FEIBA have been described to be potentially useful in dabigatran-related bleeding [3–5]. rFVIIA was chosen in this case rather than FEIBA due to rapid local availability. The haematoma was successfully evacuated with good haemostasis. The patient was extubated 24 h later and returned home after a period of rehabilitation. Thrombin generation assays pre-rFVIIa treatment displayed an increased lag time and time to peak thrombin generation with markedly reduced peak thrombin generation and endogenous thrombin potential (ETP) of 59 and 43 % of controls. In post-administration of rFVIIa, there was a partial correction of all parameters with peak thrombin generation and ETP correcting to 83 and 75 %, respectively (Fig. 1). Dabigatran level 2 h post-rFVIIa (12 h post-ingestion) decreased to sub-therapeutic level at 90 ng/ml [1], most likely due to renal elimination in a time-dependent manner. APTT normalized but TCT remained prolonged at 152 s, consistent with the ongoing presence of dabigatran at sub-therapeutic level [6]. Recombinant FVIIa has been proposed as a potential therapeutic agent in cases of dabigatran-related bleeding based upon in vivo animal models and in vitro data from healthy volunteers with inconclusive results [3, 4, 7, 8]. This case demonstrates the potential efficacy of rFVIIa in managing dabigatran-related critical organ bleeding by restoring the patient’s haemostatic potential as demonstrated by the enhanced thrombin generation. Whilst 90 μg/kg dose achieved the desired haemostatic effect in this case, thrombin generation was not fully restored; hence, the optimal dosing and efficacy are still uncertain, especially for supra-therapeutic drug levels [4]. In conclusion, this case highlights that the successful outcome was the result of timely neurosurgical intervention, combined the use of rFVIIa as a haemostatic agent. Further studies, however, are required to determine the optimal role and dosing of rFVIIa in dabigatran-related bleeding. K. T. Htun (*) : J. McFadyen :H. A. Tran Department of Haematology, Alfred Health, 55 Commercial Road, Melbourne, Victoria 3004, Australia e-mail: K.Htun@alfred.org.au DOI 10.1007/s00277-014-2039-7 Ann Hematol (2014) 93:1785 1786

Targeting activated platelets: A unique and potentially universal approach for cancer imaging

Rationale The early detection of primary tumours and metastatic disease is vital for successful therapy and is contingent upon highly specific molecular markers and sensitive, non-invasive imaging techniques. We hypothesized that the accumulation of activated platelets within tumours is a general phenomenon and thus represents a novel means for the molecular imaging of cancer. Here we investigate a unique single chain antibody (scFv), which specifically targets activated platelets, as a novel biotechnological tool for molecular imaging of cancer. Methods The scFvGPIIb/IIIa, which binds specifically to the activated form of the platelet integrin receptor GPIIb/IIIa present on activated platelets, was conjugated to either Cy7, 64Cu or ultrasound-enhancing microbubbles. Using the Cy7 labelled scFvGPIIb/IIIa, fluorescence imaging was performed in mice bearing four different human tumour xenograft models; SKBr3, MDA-MB-231, Ramos and HT-1080 cells. Molecular imaging via PET and ultrasound was performed using the scFvGPIIb/IIIa-64Cu and scFvGPIIb/IIIa-microbubbles, respectively, to further confirm specific targeting of scFvGPIIb/IIIa to activated platelets in the tumour stroma. Results Using scFvGPIIb/IIIa we successfully showed specific targeting of activated platelets within the microenvironment of human tumour xenografts models via three different molecular imaging modalities. The presence of platelets within the tumour microenvironment, and as such their relevance as a molecular target epitope in cancer was further confirmed via immunofluorescence of human tumour sections of various cancer types, thus validating the translational importance of our novel approach to human disease. Conclusion Our study provides proof of concept for imaging and localization of tumours by molecular targeting activated platelets. We illustrate the utility of a unique scFv as a versatile biotechnological tool which can be conjugated to various contrast agents for molecular imaging of cancer using three different imaging modalities. These findings warrant further development of this activated platelet specific scFvGPIIb/IIIa, potentially as a universal marker for cancer diagnosis and ultimately for drug delivery in an innovative theranostic approach.