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

Mid-Miocene cooling and the extinction of tundra in continental Antarctica

A major obstacle in understanding the evolution of Cenozoic climate has been the lack of well dated terrestrial evidence from high-latitude, glaciated regions. Here, we report the discovery of exceptionally well preserved fossils of lacustrine and terrestrial organisms from the McMurdo Dry Valleys sector of the Transantarctic Mountains for which we have established a precise radiometric chronology. The fossils, which include diatoms, palynomorphs, mosses, ostracodes, and insects, represent the last vestige of a tundra community that inhabited the mountains before stepped cooling that first brought a full polar climate to Antarctica. Paleoecological analyses, 40Ar/39Ar analyses of associated ash fall, and climate inferences from glaciological modeling together suggest that mean summer temperatures in the region cooled by at least 8°C between 14.07 ± 0.05 Ma and 13.85 ± 0.03 Ma. These results provide novel constraints for the timing and amplitude of middle-Miocene cooling in Antarctica and reveal the ecological legacy of this global climate transition

Spectral Analysis of the Lower Eocene Wilkins Peak Member, Green River Formation, Wyoming: Support for Milankovitch Cyclicity

This study is the first to employ spectral analysis to examine meter-scale sedimentary cyclicity in the Wilkins Peak Member of the lower Eocene Green River Formation of Wyoming. Generally regarded as the classic example for orbital forcing of lacustrine sediments at eccentricity and precession time scales, this long-standing interpretation was recently contested, with a much shorter duration (≤ 10 ky) inferred for the dominant cyclicity. Earlier work lacked adequate age control or spectral analysis or both. Our analysis is based upon an evaluation in the frequency domain of oil-yield values from four boreholes, accuracy estimation for suggested orbital interpretations, and comparison to independent geochronology. Cored intervals 266–364 m thick represent a span of 1.2–1.7 m.y., with temporal resolution of ∼ 3–5 ky (∼ 1 m) for oil-yield values. Variations in spectral power with depth within the original records are interpreted to reflect changes in the rate of sediment accumulation. These changes are corrected prior to testing the orbital forcing hypothesis by using two methods: 1) a minimal adjustment (three segments) accounting for the dominant changes of spectral frequency with depth; and 2) correlating the published definitions of precessional cycles in these records to a 21 ky cosine curve. Orbital age models resulting from the two tuning methods are compared to available chronology and the tuned records are tested for the expected spectral peaks from orbitally forced records. We conclude that the dominant cyclicity of the Wilkins Peak Member is orbitally forced. Orbital age models overlap 40Ar/39Ar ages and inferred periods include long and short eccentricity, weak obliquity and precession. Eccentricity is resolved in the analyzed records but the expected ∼ 95 and ∼ 125 ky periods are not resolved, controlling the range of possible tuning periods and the accuracy of orbital age models. Sub-Milankovitch variability exists and can be resolved to a minimum period of ∼ 3–5 ky by the analyzed records. However, it cannot be characterized fully with the available chronology or by the previously calculated mean cycle duration

Eocene Calibration of Geomagnetic Polarity Time Scale Reevaluated: Evidence from the Green River Formation of Wyoming

We reevaluate the Eocene geomagnetic polarity time scale on the basis of single-crystal 40Ar/39Ar ages for air-fall tuffs from the Wilkins Peak Member of the Green River Formation of Wyoming. Tuff 6 is dated as 49.1 ± 0.2 Ma, and tuff 3 is dated as 50.4 ± 0.3 Ma (maximum estimate). When combined with published magnetostratigraphic constraints, these age determinations suggest that the currently accepted age of chron C22r is 1.5–2.5 m.y. too old, which supports a significantly longer duration for the early Eocene, for the early Eocene climatic optimum, and the Wasatchian North American Land Mammal Age

A strategy for cross-calibrating U–Pb chronology and astrochronology of sedimentary sequences: An example from the Green River Formation, Wyoming, USA

Astronomical calibration of the geological timescale has been limited until recently by the precision and accuracy of radioisotopic dates, especially for pre-Neogene records. Uncertainties for radioisotopic dates of older strata were typically much larger than a single precessional cycle, and dates were often sparse, leading to the practice of orbital tuning of cyclic strata in order to astronomically calibrate the desired interval. Ideally, in order to test the assumptions of astronomical calibration with geochronology, it is necessary that the precision of radioisotopic dates be comparable to the period of the cycle being tested. The new U–Pb CA-TIMS (chemical abrasion–thermal ionization mass spectrometry) zircon dates reported here conform to this precision requirement, with 2σ analytical uncertainties from ±11000 to ±52 000 years for seven volcanic ashes from the Wilkins Peak Member of the Green River Formation. The zircon dates have simple distributions with few outliers and allow accurate estimations of the eruption ages with potential inaccuracies of less than precessional cycle. The Eocene Green River Formation (Wyoming, USA) has long been recognized as a record of cyclicly- deposited lacustrine sediments, and the abundant intercalated volcanic ashes make it a suitable place to test new approaches to astronomical calibration of cyclic strata. The abundance of different types of marker beds, including tuffs that are intercalated with the sedimentary cycles, guarantee an unambiguous correlation between sampling locations of dated tuffs on the margins of the basin and the basin center where the cyclicity is best developed, thus reducing any stratigraphic uncertainties to a fraction of (hypothesized) precession cycle. Tuning-based orbital age models, accepted by the previous geochronology, significantly deviate from the new geochronology, whereas a previously rejected model that assumes a short eccentricity period of 125 ky is now allowed. In order to test possible explanations for the apparent 125 ky period, such as changes in orbital periods, or gaps in the sedimentary record, we present an iterative strategy to select future ashes for dating such that the astronomical calibration/testing is optimized. We iteratively contrast two ad-hoc age models that bracket the linear interpolation between the dated ashes. The optimal intervals for further dating are located where the deviations between the models exceed our reported uncertainties. We propose that the iterative approach described here should become the standard for establishing a rigorous orbital calibration of the stratigraphic record where sufficient ashes exist

Evidence for a cytoplasmic proplatelet promoting factor that triggers platelet production

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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