Kristina M. Haley

The PAK system links Rho GTPase signaling to thrombin-mediated platelet activation

Regulation of the platelet actin cytoskeleton by the Rho family of small GTPases is essential for the proper maintenance of hemostasis. However, little is known about how intracellular platelet activation from Rho GTPase family members, including Rac, Cdc42, and Rho, translate into changes in platelet actin structures. To better understand how Rho family GTPases coordinate platelet activation, we identified platelet proteins associated with Rac1, a Rho GTPase family member, and actin regulatory protein essential for platelet hemostatic function. Mass spectrometry analysis revealed that upon platelet activation with thrombin, Rac1 associates with a set of effectors of the p21-activated kinases (PAKs), including GIT1, βPIX, and guanine nucleotide exchange factor GEFH1. Platelet activation by thrombin triggered the PAK-dependent phosphorylation of GIT1, GEFH1, and other PAK effectors, including LIMK1 and Merlin. PAK was also required for the thrombin-mediated activation of the MEK/ERK pathway, Akt, calcium signaling, and phosphatidylserine (PS) exposure. Inhibition of PAK signaling prevented thrombin-induced platelet aggregation and blocked platelet focal adhesion and lamellipodia formation in response to thrombin. Together, these results demonstrate that the PAK signaling system is a key orchestrator of platelet actin dynamics, linking Rho GTPase activation downstream of thrombin stimulation to PAK effector function, MAP kinase activation, calcium signaling, and PS exposure in platelets.

p21 Activated Kinase Signaling Coordinates Glycoprotein Receptor VI–Mediated Platelet Aggregation, Lamellipodia Formation, and Aggregate Stability Under Shear

Objective—Rho GTPase proteins play a central role in regulating the dynamics of the platelet actin cytoskeleton. Yet, little is known regarding how Rho GTPase activation coordinates platelet activation and function. In this study, we aimed to characterize the role of the Rho GTPase effector, p21 activated kinase (PAK), in platelet activation, lamellipodia formation, and aggregate formation under shear. Approach and Results—Stimulation of platelets with the glycoprotein receptor VI agonist, collagen-related peptide, rapidly activated PAK in a time course preceding phosphorylation of PAK substrates, LIM domain kinase LIMK1 and the MAPK/ERK kinase MEK, and the subsequent activation of MAPKs and Akt. Pharmacological inhibitors of PAK blocked signaling events downstream of PAK and prevented platelet secretion as well as platelet aggregation in response to collagen-related peptide. PAK inhibitors also prevented PAK activation and platelet spreading on collagen surfaces. PAK was also required for the formation of platelet aggregates and to maintain aggregate stability under physiological shear flow conditions. Conclusions—These results suggest that PAK serves as an orchestrator of platelet functional responses after activation downstream of the platelet collagen receptor, glycoprotein receptor VI.

Thrombopoietin Receptor Agonist Use in Children: Data From the Pediatric ITP Consortium of North America ICON2 Study

Data on second‐line treatment options for pediatric patients with immune thrombocytopenia (ITP) are limited. Thrombopoietin receptor agonists (TPO‐RA) provide a nonimmunosuppressive option for children who require an increased platelet count.

Do circulating tumor cells play a role in coagulation and thrombosis

Cancer induces a hypercoagulable state, and patients with cancer who suffer a thrombotic event have a worse prognosis than those who do not. Recurrent pathologic thrombi in patients with cancer are clinically managed with anticoagulant medications; however, anticoagulant prophylaxis is not routinely prescribed owing to a complex variety of patient and diagnosis related factors. Early identification of patients at risk for cancer-associated thrombosis would allow for personalization of anticoagulant prophylaxis and likely reduce morbidity and mortality for many cancers. The environment in which a thrombosis develops in a patient with cancer is complex and unique from patients without cancer, which creates therapeutic challenges but may also provide targets for the development of clinical assays in this context. Circulating tumor cells (CTCs) may play a role in the association between cancer and thrombosis. Cancer metastasis, the leading cause of cancer-related deaths, is facilitated by the hematogenous spread of CTCs, and CTCs accompany metastatic disease across all major types of carcinomas. The role of CTCs in the pathogenesis of thrombosis has not been studied due to the previous difficulty in identifying these rare cells, but the interaction between these circulating cells and the coagulation system is an area of study that demands attention. The development of CTC detection platforms presents a new tool by which to characterize the role for CTCs in cancer-related hypercoagulability. In addition, this area of study presents a new avenue for assessing the risk of cancer-associated thrombosis and represents a potential tool for predicting which patients may benefit from anticoagulant prophylaxis. In this review, we will discuss the evidence in support of CTC induced hypercoagulability, and highlight areas where CTC-detection platforms may provide prognostic insight into the risk of developing thrombosis for patients with cancer.

Assessment of neonatal platelet adhesion, activation, and aggregation

Essentials Assays are needed to aid in the diagnosis of platelet dysfunction in the neonatal population. We developed small‐volume assays to assess neonatal platelet activation and aggregation. Compared to adult platelets, neonatal platelet activation and secretion was reduced. The extent of neonatal and adult platelet adhesion and aggregate formation were similar.

Neonatal platelets: mediators of primary hemostasis in the developing hemostatic system

The human hemostatic system is developmentally regulated, resulting in qualitative and quantitative differences in the mediators of primary and secondary hemostasis as well as fibrinolysis in neonates and infants. Although gestational age–related differences in coagulation factor levels occur, the existence of a unique neonatal platelet phenotype remains controversial. Complicated by difficulties in obtaining adequate neonatal blood volumes with which to perform functional assays, ambiguity surrounds the characterization of neonatal platelets. Thus, much of the current knowledge of neonatal platelet function has been based on studies from cord blood samples. Studies suggest that cord blood–derived platelets, as a surrogate for neonatal platelets, are hypofunctional when compared with adult platelets. This relative platelet dysfunction, combined with a propensity toward thrombocytopenia in the neonatal intensive care unit population, creates a clinical conundrum regarding the appropriate administration of platelet transfusions. This review provides an appraisal of the distinct functional phenotype of neonatal platelets. Neonatal platelet transfusion practices and the impact of the relatively hypofunctional neonatal platelet on those practices will be considered.Pediatric Research (2014); 76 3, 230–237. doi:10.1038/pr.2014.87

Congenital Hemolytic Anemia.

Red blood cell (RBC) destruction can be secondary to intrinsic disorders of the RBC or to extrinsic causes. In the congenital hemolytic anemias, intrinsic RBC enzyme, RBC membrane, and hemoglobin disorders result in hemolysis. The typical clinical presentation is a patient with pallor, anemia, jaundice, and often splenomegaly. The laboratory features include anemia, hyperbilirubinemia, and reticulocytosis. For some congenital hemolytic anemias, splenectomy is curative. However, in other diseases, avoidance of drugs and toxins is the best therapy. Supportive care with transfusions are also mainstays of therapy. Chronic hemolysis often results in the formation of gallstones, and cholecystectomy is often indicated.

Fatal carboplatin-induced immune hemolytic anemia in a child with a brain tumor.

Drug-induced immune hemolytic anemia (DIIHA) is an uncommon side effect of pharmacologic intervention. A rare mediator of DIIHA, carboplatin is an agent used to treat many pediatric cancers. We describe here, the first case of fatal carboplatin induced DIIHA in a pediatric patient and a brief review of the literature. Our patient developed acute onset of multi-organ failure with evidence of complement activation, secondary to a drug induced red cell antibody. Early recognition of the systemic insult associated with carboplatin induced hemolytic anemia may allow for future affected patients to receive plasmapheresis, a potentially effective therapy.

Physician decision making in selection of second-line treatments in immune thrombocytopenia in children

Immune thrombocytopenia (ITP) is an acquired autoimmune bleeding disorder which presents with isolated thrombocytopenia and risk of hemorrhage. While most children with ITP promptly recover with or without drug therapy, ITP is persistent or chronic in others. When needed, how to select second‐line therapies is not clear. ICON1, conducted within the Pediatric ITP Consortium of North America (ICON), is a prospective, observational, longitudinal cohort study of 120 children from 21 centers starting second‐line treatments for ITP which examined treatment decisions. Treating physicians reported reasons for selecting therapies, ranking the top three. In a propensity weighted model, the most important factors were patient/parental preference (53%) and treatment‐related factors: side effect profile (58%), long‐term toxicity (54%), ease of administration (46%), possibility of remission (45%), and perceived efficacy (30%). Physician, health system, and clinical factors rarely influenced decision‐making. Patient/parent preferences were selected as reasons more often in chronic ITP (85.7%) than in newly diagnosed (0%) or persistent ITP (14.3%, P = .003). Splenectomy and rituximab were chosen for the possibility of inducing long‐term remission (P < .001). Oral agents, such as eltrombopag and immunosuppressants, were chosen for ease of administration and expected adherence (P < .001). Physicians chose rituximab in patients with lower expected adherence (P = .017). Treatment choice showed some physician and treatment center bias. This study illustrates the complexity and many factors involved in decision‐making in selecting second‐line ITP treatments, given the absence of comparative trials. It highlights shared decision‐making and the need for well‐conducted, comparative effectiveness studies to allow for informed discussion between patients and clinicians.

neonatal venous thromboembolism

Neonates are the pediatric population at highest risk for development of venous thromboembolism (VTE), and the incidence of VTE in the neonatal population is increasing. This is especially true in the critically ill population. Several large studies indicate that the incidence of neonatal VTE is up almost threefold in the last two decades. Central lines, fluid fluctuations, sepsis, liver dysfunction, and inflammation contribute to the risk profile for VTE development in ill neonates. In addition, the neonatal hemostatic system is different from that of older children and adults. Platelet function, pro- and anticoagulant proteins concentrations, and fibrinolytic pathway protein concentrations are developmentally regulated and generate a hemostatic homeostasis that is unique to the neonatal time period. The clinical picture of a critically ill neonate combined with the physiologically distinct neonatal hemostatic system easily fulfills the criteria for Virchow’s triad with venous stasis, hypercoagulability, and endothelial injury and puts the neonatal patient at risk for VTE development. The presentation of a VTE in a neonate is similar to that of older children or adults and is dependent upon location of the VTE. Ultrasound is the most common diagnostic tool employed in identifying neonatal VTE, but relatively small vessels of the neonate as well as frequent low pulse pressure can make ultrasound less reliable. The diagnosis of a thrombophilic disorder in the neonatal population is unlikely to change management or outcome, and the role of thrombophilia testing in this population requires further study. Treatment of neonatal VTE is aimed at reducing VTE-associated morbidity and mortality. Recommendations for treating, though, cannot be extrapolated from guidelines for older children or adults. Neonates are at risk for bleeding complications, particularly younger neonates with more fragile intracranial vessels. Developmental alterations in the coagulation proteins as well as unique pharmacokinetics must also be taken into consideration when recommending VTE treatment. In this review, epidemiology of neonatal VTE, pathophysiology of neonatal VTE with particular attention to the developmental hemostatic system, diagnostic evaluations of neonatal VTE, and treatment guidelines for neonatal VTE will be reviewed.