The incredible journey: From megakaryocyte development to platelet formation

Circulating blood platelets are specialized cells that prevent bleeding and minimize blood vessel injury. Large progenitor cells in the bone marrow called megakaryocytes (MKs) are the source of platelets. MKs release platelets through a series of fascinating cell biological events. During maturation, they become polyploid and accumulate massive amounts of protein and membrane. Then, in a cytoskeletal-driven process, they extend long branching processes, designated proplatelets, into sinusoidal blood vessels where they undergo fission to release platelets. Given the need for platelets in many pathological situations, understanding how this process occurs is an active area of research with important clinical applications

Pathophysiology of plasma hypercoagulability in thrombosis

Blood coagulation abnormalities are the leading cause of death world-wide. Elevated procoagulant factor levels (hypercoagulability) have been correlated with increased thrombin generation and increased risk of arterial and venous thrombosis. This dissertation explores the role of hypercoagulability on various aspects of coagulation and thrombosis in vitro and in vivo. Thrombin generation tests are increasingly being employed as a high throughput, global measure of procoagulant activity. Thrombin generation was measured using calibrated automated thrombography (CAT) in platelet-free plasma (PFP) and platelet-rich plasma (PRP). The relative sensitivity of CAT parameters to elevated factors XI, IX, VIII, X, and prothrombin was: PFP initiated with 1 pM tissue factor (TF) > PFP initiated with 5 pM TF > PRP initiated with 1 pM TF. Monitoring the peak height following initiation with 1 pM TF in PFP was most likely to detect hypercoagulability (increased procoagulant factors). Epidemiologic studies have correlated elevated plasma fibrinogen (hyperfibrinogenemia) with risk of arterial and venous thrombosis. However, it is unknown whether hyperfibrinogenemia is a biomarker of disease or causative in the etiology. In mice, hyperfibrinogenemia significantly shortened the time to occlusion (TTO) after FeCl3 injury to the saphenous vein and carotid artery. Hyperfibrinogenemia increased thrombus fibrin content, promoted faster fibrin formation, and increased fibrin network density, strength, and stability and increased thrombus thrombolysis resistance in vivo. These data indicate hyperfibrinogenemia directly promotes thrombosis and thrombolysis resistance via enhanced fibrin formation and stability. Studies have correlated elevated plasma factor VIII (FVIII) with thrombosis. However, like hyperfibrinogenemia, it is unclear whether elevated FVIII is a biomarker or causative agent. In mice, elevated FVIII had no effect on 3-minute FeCl3 carotid artery injury, but shortened the TTO after 2-minute injury. Additionally, elevated FVIII increased circulating thrombin-antithrombin complexes and stabilized clots after 2- but not 3-minute FeCl3 injury. In vitro, elevated FVIII increased thrombin generation and accelerated platelet aggregation only when initiated by low TF. These results demonstrate dependence of FVIII thrombogenicity on extent of vascular injury. These findings provide a better understanding of how plasma hypercoagulability impacts thrombogenesis. Specifically, these data suggest causative yet differential roles for hyperfibrinogenemia and elevated FVIII in thrombosis

Procoagulant Activity in Hemostasis and Thrombosis: Virchowʼs Triad Revisited

Virchow’s triad is traditionally invoked to explain pathophysiologic mechanisms leading to thrombosis, alleging concerted roles for abnormalities in blood composition, vessel wall components, and blood flow in the development of arterial and venous thrombosis. Given the tissue-specific bleeding observed in hemophilia patients, it may be instructive to consider the principles of Virchow’s triad when investigating mechanisms operant in hemostatic disorders as well. Blood composition (the function of circulating blood cells and plasma proteins) is the most well-studied component of the triad. For example, increased levels of plasma procoagulant proteins such as prothrombin and fibrinogen are established risk factors for thrombosis, whereas deficiencies in plasma factors VIII and IX result in bleeding (hemophilia A and B, respectively). Vessel wall (cellular) components contribute adhesion molecules that recruit circulating leukocytes and platelets to sites of vascular damage, tissue factor, which provides a procoagulant signal of vascular breach, and a surface upon which coagulation complexes are assembled. Blood flow is often characterized by two key variables: shear rate and shear stress. Shear rate affects several aspects of coagulation, including transport rates of platelets and plasma proteins to and from the injury site, platelet activation, and the kinetics of fibrin monomer formation and polymerization. Shear stress modulates adhesion rates of platelets and expression of adhesion molecules and procoagulant activity on endothelial cells lining the blood vessels. That no one abnormality in any component of Virchow’s triad fully predicts coagulopathy a priori suggests coagulopathies are complex, multifactorial and interactive. In this review, we focus on contributions of blood composition, vascular cells, and blood flow to hemostasis and thrombosis, and suggests cross-talk among the three components of Virchow’s triad is necessary for hemostasis and determines propensity for thrombosis or bleeding. Investigative models that permit interplay among these components are necessary to understand the operant pathophysiology, and effectively treat and prevent thrombotic and bleeding disorders

Recombinant factor VIIa analog NN1731 (V158D/E296V/M298Q-FVIIa) enhances fibrin formation, structure and stability in lipidated hemophilic plasma

Introduction—The bypassing agent recombinant factor VIIa (rFVIIa) is efficacious in treating bleeding in hemophilia patients with inhibitors. Efforts have focused on the rational engineering of rFVIIa variants with increased hemostatic potential. One rFVIIa analog (V158D/E296V/M298QFVIIa, NN1731) improves thrombin generation and clotting in purified systems, whole blood from hemophilic patients and factor VIII-deficient mice. Methods—We used calibrated automated thrombography and plasma clotting assays to compare effects of bypassing agents (rFVIIa, NN1731) on hemophilic clot formation, structure, and ability to resist fibrinolysis. Results—Both rFVIIa and NN1731 shortened the clotting onset and increased the maximum rate of fibrin formation and fibrin network density in hemophilic plasma clots. In the presence of tissue plasminogen activator, both rFVIIa and NN1731 shortened the time to peak turbidity (TTPeaktPA) and increased the area under the clot formation curve (AUCtPA). Phospholipids increased both rFVIIa and NN1731 activity in a lipid concentration-dependent manner. Estimated geometric mean concentrations of rFVIIa and NN1731 producing similar onset, rate, TTPeaktPA, and AUCtPA as seen with 100% factors VIII and IX were: 24.5, 74.3, 29.7, and 37.1 nM rFVIIa, and 8.6, 31.2, 9.0, and 11.3 nM NN1731, respectively. In each case, the NN1731 concentration was significantly lower than rFVIIa. Conclusions—These findings suggest that like rFVIIa, NN1731 improves the formation, structure, and stability of hemophilic clots. Higher lipid concentrations may facilitate assessment of both rFVIIa and NN1731 activity. NN1731 appears likely to support rapid clot formation in tissues with high endogenous fibrinolytic activity

Effects of tissue factor, thrombomodulin and elevated clotting factor levels on thrombin generation in the calibrated automated thrombogram

Elevated procoagulant levels have been correlated with increased thrombin generation in vitro and with increased venous thromboembolism (VTE) risk in epidemiological studies. hrombin generation tests are increasingly being employed as a high throughput method to provide a global measure of procoagulant activity in plasma samples. The objective of this study was to distinguish the effects of assay conditions [tissue factor (TF), thrombomodulin, platelets/lipids] and factor levels on thrombin generation parameters, and determine the conditions and parameters with the highest sensitivity and specificity for detecting elevated factor levels. Thrombin generation was measured using calibrated automated thrombography (CAT) in corn trypsin inhibitor (CTI)-treated platelet-free plasma (PFP) and plateletrich plasma (PRP). Statistical analysis was performed using logarithms of observed values with analysis of variance that accounted for experiment and treatment. he relative sensitivity of lag time (LT), time to peak (TTP), peak height and endogenous thrombin potential (ETP) to elevated factors XI, IX,VIII, X, and prothrombin was as follows: PFP initiated with 1 pM TF > PFP initiated with 5 pM TF > PRP initiated with 1 pM TF. For all conditions, inclusion of thrombomodulin prolonged the LT and decreased the peak and ETP; however, addition of thrombomodulin did not increase the ability of CAT to detect elevated levels of individual procoagulant factors. In conclusion, CAT conditions differentially affected the sensitivity of thrombin generation to elevated factor levels. Monitoring the peak height and/ or ETP following initiation of clotting in PFP with 1 pM TF was most likely to detect hypercoagulability due to increased procoagulant factor levels

Venous thromboembolism research priorities: A scientific statement from the American Heart Association and the International Society on Thrombosis and Haemostasis

Venous thromboembolism (VTE) is a major cause of morbidity and mortality. The impact of the Surgeon General’s Call to Action in 2008 has been lower than expected given the public health impact of this disease. This scientific statement highlights future research priorities in VTE, developed by experts and a crowdsourcing survey across 16 scientific organizations. At the fundamental research level (T0), researchers need to identify pathobiologic causative mechanisms for the 50% of patients with unprovoked VTE and better understand mechanisms that differentiate hemostasis from thrombosis. At the human level (T1), new methods for diagnosing, treating, and preventing VTE will allow tailoring of diagnostic and therapeutic approaches to individuals. At the patient level (T2), research efforts are required to understand how foundational evidence impacts care of patients (eg, biomarkers). New treatments, such as catheter‐based therapies, require further testing to identify which patients are most likely to experience benefit. At the practice level (T3), translating evidence into practice remains challenging. Areas of overuse and underuse will require evidence‐based tools to improve care delivery. At the community and population level (T4), public awareness campaigns need thorough impact assessment. Large population‐based cohort studies can elucidate the biologic and environmental underpinings of VTE and its complications. To achieve these goals, funding agencies and training programs must support a new generation of scientists and clinicians who work in multidisciplinary teams to solve the pressing public health problem of VTE.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/156163/2/rth212373_am.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/156163/1/rth212373.pd

Detection of endogenous tissue factor levels in plasma using the calibrated automated thrombogram assay

The calibrated automated thrombogram (CAT) assay measures thrombin generation in plasma

T granules in human platelets function in TLR9 organization and signaling

Human and murine platelets (PLTs) variably express toll-like receptors (TLRs), which link the innate and adaptive immune responses during infectious inflammation and atherosclerotic vascular disease. In this paper, we show that the TLR9 transcript is specifically up-regulated during pro-PLT production and is distributed to a novel electron-dense tubular system-related compartment we have named the T granule. TLR9 colocalizes with protein disulfide isomerase and is associated with either VAMP 7 or VAMP 8, which regulates its distribution in PLTs on contact activation (spreading). Preincubation of PLTs with type IV collagen specifically increased TLR9 and CD62P surface expression and augmented oligodeoxynucleotide (ODN) sequestration and PLT clumping upon addition of bacterial/viral ODNs. Collectively, this paper (a) tracks TLR9 to a new intracellular compartment in PLTs and (b) describes a novel mechanism of TLR9 organization and signaling in human PLTs

Scalable Generation of Universal Platelets from Human Induced Pluripotent Stem Cells

Summary Human induced pluripotent stem cells (iPSCs) provide a potentially replenishable source for the production of transfusable platelets. Here, we describe a method to generate megakaryocytes (MKs) and functional platelets from iPSCs in a scalable manner under serum/feeder-free conditions. The method also permits the cryopreservation of MK progenitors, enabling a rapid “surge” capacity when large numbers of platelets are needed. Ultrastructural/morphological analyses show no major differences between iPSC platelets and human blood platelets. iPSC platelets form aggregates, lamellipodia, and filopodia after activation and circulate in macrophage-depleted animals and incorporate into developing mouse thrombi in a manner identical to human platelets. By knocking out the β2-microglobulin gene, we have generated platelets that are negative for the major histocompatibility antigens. The scalable generation of HLA-ABC-negative platelets from a renewable cell source represents an important step toward generating universal platelets for transfusion as well as a potential strategy for the management of platelet refractoriness