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With the dramatic expansion of the biomedical knowledge base and increasing demands for evidence-based medicine, the role of the clinician-scientist is becoming increasingly important. In orthopaedic surgery, clinician-scientists are at the forefront of translational efforts to address the growing burden of musculoskeletal disease, yet MD-PhD trained investigators have historically been underrepresented in this field. Here, we examine the trend, over time, of MD-PhD graduates pursuing orthopaedic surgery, compared with other specialties. Survey data from the 2018 Association of American Medical Colleges National MD-PhD Program Outcomes Study, including data on 4,647 individuals who had completed residency training and 2,124 who were still in training, were reanalyzed. Numbers, proportions, workplace choice, and percent research effort of MD-PhD graduates completing orthopaedic surgery were compared with other surgical and nonsurgical specialties. Trends over time were analyzed by linear regression. While a decreasing proportion of MD-PhD graduates completed internal medicine training, just 1.1% of MD-PhD graduates completed orthopaedic surgery training, lower than that of all other surgical specialties. The proportion of MD-PhD graduates completing orthopaedic surgery has not increased over time and was mirrored in MD-PhD residents still in training. Though MD-PhDs are increasingly choosing to pursue "nontraditional" specialties, they remain underrepresented in orthopaedic surgery, compared with other clinical disciplines. Thus, there exists an opportunity to encourage MD-PhD graduates to pursue careers in orthopaedic surgery, to supplement the existing intellectual capital in the orthopaedic science workforce. This, along with other strategies to support all orthopaedic surgeon-scientists, will ultimately advance the care of musculoskeletal diseases.

Rebalancing the hemostatic system by targeting endogenous anticoagulant pathways, like the protein C (PC) system, is being tested as a means of improving hemostasis in patients with hemophilia. Recent intravital studies of hemostasis demonstrated that, in some vascular contexts, thrombin activity is sequestered in the extravascular compartment. These findings raise important questions about the context-dependent contribution of activated PC (APC) to the hemostatic response, because PC activation occurs on the surface of endothelial cells. We used a combination of pharmacologic, genetic, imaging, and computational approaches to examine the relationships among thrombin spatial distribution, PC activation, and APC anticoagulant function. We found that inhibition of APC activity, in mice either harboring the factor V Leiden mutation or infused with an APC-blocking antibody, significantly enhanced fibrin formation and platelet activation in a microvascular injury model, consistent with the role of APC as an anticoagulant. In contrast, inhibition of APC activity had no effect on hemostasis after penetrating injury of the mouse jugular vein. Computational studies showed that differences in blood velocity, injury size, and vessel geometry determine the localization of thrombin generation and, consequently, the extent of PC activation. Computational predictions were tested in vivo and showed that when thrombin generation occurred intravascularly, without penetration of the vessel wall, inhibition of APC significantly increased fibrin formation in the jugular vein. Together, these studies show the importance of thrombin spatial distribution in determining PC activation during hemostasis and thrombosis.

BACKGROUND: Tissue factor pathway inhibitor (TFPI) is an essential regulator of coagulation, limiting thrombin generation and preventing thrombosis. In humans and mice, TFPIα is the sole isoform present in platelets. OBJECTIVE: Here, we asked whether TFPIα, because of its release from platelets at sites of injury, has a unique role in limiting the hemostatic response. METHODS: TFPIα-mutant (Tfpi(Δα/Δα)) mice were generated by introducing a stop codon in the C-terminus. Platelet accumulation, platelet activation, and fibrin accumulation were measured following penetrating injuries in the jugular vein and cremaster muscle arterioles, and imaged by fluorescence and scanning electron microscopy. Time to bleeding cessation was recorded in the jugular vein studies. RESULTS: Tfpi(Δα/Δα) mice were viable and fertile. Plasma TFPI levels were normal in the Tfpi(Δα/Δα) mice, no TFPI protein or activity was present in their platelets and thrombin-antithrombin complex levels were indistinguishable from Tfpi(+/+) littermates. There was a small, but statistically significant reduction in the time to bleeding cessation following jugular vein puncture injury in the Tfpi(Δα/Δα) mice, but no measurable changes in platelet or fibrin accumulation or in hemostatic plug architecture following injury of the micro- or macrovasculature. CONCLUSION: Loss of TFPIα expression does not produce a global prothrombotic state in mice. Platelet TFPIα is expected to be released or displayed in a focal manner at the site of injury, potentially accumulating to high concentrations in the narrow gaps between platelets. If so, the data from the vascular injury models studied here indicate this is not essential for a normal hemostatic response in mice.

Hemostasis is an innate protective mechanism that plays a central role in maintaining the homeostasis of the vascular system during vascular injury. Studying this essential physiological process is often challenged by the difficulty of modeling and probing the complex dynamics of hemostatic responses in the native context of human blood vessels. To address this major challenge, this paper describes a microengineering approach for in vitro modeling of hemostasis. Our microphysiological model replicates the living endothelium, multilayered microarchitecture, and procoagulant activity of human blood vessels, and is also equipped with a microneedle that can be actuated with spatial precision to simulate penetrating vascular injuries. The system recapitulates key features of the hemostatic response to acute vascular injury as observed in vivo, including i) thrombin-driven accumulation of platelets and fibrin, ii) formation of a platelet- and fibrin-rich hemostatic plug that halts blood loss, and iii) matrix deformation driven by platelet contraction for wound closure. Moreover, the potential use of this model for drug testing applications is demonstrated by evaluating the effects of anticoagulants and antiplatelet agents that are in current clinical use. The vascular injury-on-a-chip may serve as an enabling platform for preclinical investigation of hematological disorders and emerging therapeutic approaches against them.

Platelets are best known for their vasoprotective responses to injury and inflammation. Here, we have asked whether they also support vascular integrity when neither injury nor inflammation is present. Changes in vascular barrier function in dermal and meningeal vessels were measured in real time in mouse models using the differential extravasation of fluorescent tracers as a biomarker. Severe thrombocytopenia produced by two distinct methods caused increased extravasation of 40-kDa dextran from capillaries and postcapillary venules but had no effect on extravasation of 70-kDa dextran or albumin. This reduction in barrier function required more than 4 h to emerge after thrombocytopenia was established, reverting to normal as the platelet count recovered. Barrier dysfunction was also observed in mice that lacked platelet-dense granules, dense granule secretion machinery, glycoprotein (GP) VI, or the GPVI signaling effector phospholipase C (PLC) γ2. It did not occur in mice lacking α-granules, C type lectin receptor-2 (CLEC-2), or protease activated receptor 4 (PAR4). Notably, although both meningeal and dermal vessels were affected, intracerebral vessels, which are known for their tighter junctions between endothelial cells, were not. Collectively, these observations 1) highlight a role for platelets in maintaining vascular homeostasis in the absence of injury or inflammation, 2) provide a sensitive biomarker for detecting changes in platelet-dependent barrier function, 3) identify which platelet processes are required, and 4) suggest that the absence of competent platelets causes changes in the vessel wall itself, accounting for the time required for dysfunction to emerge.

Essentials Signaling by Gas6 through Tyro3/Axl/Mer receptors is essential for stable platelet aggregation. UNC2025 is a small molecule inhibitor of the Mer tyrosine kinase. UNC2025 decreases platelet activation in vitro and thrombus formation in vivo. UNC2025's anti-platelet effect is synergistic with inhibition of the ADP receptor, P2Y12. SummaryBackground Growth arrest-specific protein 6 signals through the TAM (TYRO-3–AXL–MERTK) receptor family, mediating platelet activation and thrombus formation via activation of the aggregate-stabilizing αIIbβ3 integrin. Objective To describe the antithrombotic effects mediated by UNC2025, a small-molecule MERTK tyrosine kinase inhibitor. Methods MERTK phosphorylation and downstream signaling were assessed by immunoblotting. Light transmission aggregometry, flow cytometry and microfluidic analysis were used to evaluate the impact of MERTK inhibition on platelet activation and stability of aggregates in vitro. The effects of MERTK inhibition on arterial and venous thrombosis, platelet accumulation at microvascular injury sites and tail bleeding times were determined with murine models. The effects of combined treatment with ADP–P2Y1&12 pathway antagonists and UNC2025 were also evaluated. Results and Conclusions Treatment with UNC2025 inhibited MERTK phosphorylation and downstream activation of AKT and SRC, decreased platelet activation, and protected animals from pulmonary embolism and arterial thrombosis without increasing bleeding times. The antiplatelet effect of UNC2025 was enhanced in combination with ADP–P2Y1&12 pathway antagonists, and a greater than additive effect was observed when these two agents with different mechanisms of inhibition were coadministered. TAM kinase signaling represents a potential therapeutic target, as inhibition of this axis, especially in combination with ADP–P2Y pathway antagonism, mediates decreased platelet activation, aggregate stability, and thrombus formation, with less hemorrhagic potential than current treatment strategies. The data presented here also demonstrate antithrombotic activity mediated by UNC2025, a novel translational agent, and support the development of TAM kinase inhibitors for clinical applications.

Complement plays a key role in host defense, but its dysregulation can cause autologous tissue injury. Complement activation is normally controlled by regulatory proteins, including factor H (FH) in plasma and membrane cofactor protein (MCP) on the cell surface. Mutations in FH and MCP are linked to atypical hemolytic uremic syndrome, a type of thrombotic microangiopathy (TMA) that causes renal failure. We describe here that disruption of FH function on the cell surface can also lead to disseminated complement-dependent macrovascular thrombosis. By gene targeting, we introduced a point mutation (W1206R) into murine FH that impaired its interaction with host cells but did not affect its plasma complement-regulating activity. Homozygous mutant mice carrying this mutation developed renal TMA as well as systemic thrombophilia involving large blood vessels in multiple organs, including liver, lung, spleen, and kidney. Approximately 30% of mutant mice displayed symptoms of stroke and ischemic retinopathy, and 48% died prematurely. Genetic deficiency of complement C3 and factor D prevented both the systemic thrombophilia and renal TMA phenotypes. These results demonstrate a causal relationship between complement dysregulation and systemic angiopathy and suggest that complement activation may contribute to various human thrombotic disorders involving both the micro- and macrovasculature.

Essentials Methods were developed to image the hemostatic response in mouse femoral arteries in real time. Penetrating injuries produced thrombi consisting primarily of platelets. Similar to arterioles, a core-shell architecture of platelet activation occurs in the femoral artery. Differences from arterioles included slower platelet activation and reduced thrombin dependence. SummaryBackground Intravital studies performed in the mouse microcirculation show that hemostatic thrombi formed after penetrating injuries develop a characteristic architecture in which a core of fully activated, densely packed platelets is overlaid with a shell of less activated platelets. Objective Large differences in hemodynamics and vessel wall biology distinguish arteries from arterioles. Here we asked whether these differences affect the hemostatic response and alter the impact of anticoagulants and antiplatelet agents. Methods Approaches previously developed for intravital imaging in the mouse microcirculation were adapted to the femoral artery, enabling real-time fluorescence imaging despite the markedly thicker vessel wall. Results Arterial thrombi initiated by penetrating injuries developed the core-and-shell architecture previously observed in the microcirculation. However, although platelet accumulation was greater in arterial thrombi, the kinetics of platelet activation were slower. Inhibiting platelet ADP P2Y12 receptors destabilized the shell and reduced thrombus size without affecting the core. Inhibiting thrombin with hirudin suppressed fibrin accumulation, but had little impact on thrombus size. Removing the platelet collagen receptor, glycoprotein VI, had no effect. Conclusions These results (i) demonstrate the feasibility of performing high-speed fluorescence imaging in larger vessels and (ii) highlight differences as well as similarities in the hemostatic response in the macro- and microcirculation. Similarities include the overall core-and-shell architecture. Differences include the slower kinetics of platelet activation and a smaller contribution from thrombin, which may be due in part to the greater thickness of the arterial wall and the correspondingly greater separation of tissue factor from the vessel lumen.

Hemostasis requires the tightly regulated interaction of the coagulation system, platelets, other blood cells and components of the vessel wall at a site of vascular injury. The dysregulation of this response may result in excessive bleeding if the response is impaired, and pathologic thrombosis with vessel occlusion and tissue ischemia if the response is overly robust. Extensive studies over several decades have elucidated the major molecular signaling pathways responsible for platelet activation and aggregation, and anti-thrombotic agents targeting several of these pathways are in widespread clinical use. This review will summarize more recent research examining mechanisms by which these multiple platelet signaling pathways are integrated in time and space at a site of vascular injury in vivo to produce an optimal hemostatic response.

Introduction: Vascular damage comes in many forms with the puncture wound likely the longest known to humans. Experimentally, vascular damage has typically been visualized in mouse models in which there is little bleeding. Under these conditions, damage is limited mostly to the endothelial layer lining the vessel. Visualization has varied from light to scanning electron microscopy. Interpretation has been dominated by intravital microscopy outcomes in which an initial layer of p-selection-exposed, i.e., α-granule secretion-positive, platelets is deposited in association with the damaged vessel wall and extended by the accumulation of a less activated outer layer of platelets. These results have given rise to a Core and Shell model of platelet-rich thrombus formation [Tomaiuolo et al., 2017]. Recently, new mouse models have been presented in which the vessel is punctured to create a 300 to 600 micron wound hole [Tomoiuolo et al., 2019]. Bleeding is now profuse. The puncture wound results have been interpreted within a Core and Shell model. However, two important aspects of the experimental data [Tomoiuolo et al., 2019] suggest that the existing model may not explain the actual results. First, p-selectin expression as a marker for α-granule secretion and platelet activation was present in limited areas towards the periphery of the resulting thrombus, not as well-defined Core. Second, the hemostatic thrombus when viewed at early stages, ex vivo, showed a pebbly distribution of platelet aggregates suggestive of nucleated platelet accumulation rather than the smooth layers that would follow from a Core and Shell model. We hypothesize that nucleated accumulation of platelet aggregates within the puncture hole could provide pedestals upon which localized accumulation of platelets form the infrastructure of a vaulted thrombus whose extravascular capping leads to bleeding cessation. Methods: To test the proposed hypothesis, we visualized the interior and overall structure of the forming puncture wound thrombus in full 3D at sub-platelet level resolution. To achieve this end, we took our proven serial block face scanning electron microscopy (SBF-SEM) protocols for visualizing platelet organelles in 3D [Pokrovskaya et al., 2018] and adapted them to the visualization of forming thrombi over 1000s of image. To localize samples for electron microscopy, we used in vivo antibody labeling [Tomaiuolo et al., 2019]. This approach had the added advantage of enabling correlative light microscopy mapping overall p-selectin, a marker of platelet secretion, and fibrin distributions against 3D, platelet resolution, thrombus morphology. Results: We found that a 1 min thrombus, pre-bleeding cessation, was structured about the localized accumulation of pedestal-like platelet aggregates along the sides of the puncture hole, extended and spaced along the extravascular adventitia. Subsequent pedestal extension formed a "pontoon" bridge that "capped" extravascularly the puncture hole. At 5 min, full bleeding cessation, we found that forming platelet thrombus had a Swiss cheese-like interior of vaults that were continuous with the intravascular vessel lumen and framed by columns of platelets, presumed pedestal extensions. The thrombus was sealed on the extravascular side by a platelet "cap" (Figure). As expected after bleeding cessation, red blood cells accumulated on the intravascular side of the cap. Formation of a tightly sealed cap was dependent on α-granule secretion as indicated by the effect of knockout of VAMP-8, the primary SNARE protein involved in a-granule release. Based upon morphology, vaults within the forming thrombus were lined with apparent procoagulant platelets providing a potential protected surface for coagulation factor activation. Conclusions: We conclude that bleeding cessation in a true puncture wound occurs from the extravascular side of the thrombus rather than through the formation of a platelet plug that fills the hole. We propose an alternative model of bleeding cessation in which localized platelet aggregates are the starting pedestal upon which all subsequent steps in puncture thrombus formation build, i.e., "Cap and Build". The extent to which properties differ among systems remains an open question. Tomaiuolo et al. 2017. Intervent. Cardiol. Clin. 6: 1-12. Pokrovskaya et al. 2018. Blood Adv. 2: 2947-2958 Tomaiuolo et al. 2019. Proc. Natl. Acad. Sci. USA 116:2 243-2252 Figure Disclosures No relevant conflicts of interest to declare.

In 2015, a nation-wide effort was launched to track the careers of over 10,000 MD-PhD program graduates. Data were obtained by surveys sent to alumni, inquiries sent to program directors, and searches in American Association of Medical Colleges (AAMC) databases. Here, we present an analysis of the data, focusing on the impact of sex, race, and ethnicity on career outcomes. The results show that diversity among trainees has increased since the earliest MD-PhD programs, although it still lags considerably behind the US population. Training duration, which includes time to graduation as well as time to first independent position, was similar for men and women and for minority and nonminority alumni, as were most choices of medical specialties. Regardless of minority status and sex, most survey responders reported that they are working in academia, research institutes, federal agencies, or industry. These similarities were, however, accompanied by several noteworthy differences: (a) Based on AAMC Faculty Roster data rather than survey responses, women were less likely than men to have had a full-time faculty appointment, (b) minorities who graduated after 1985 had a longer average time to degree than nonminorities, (c) fewer women and minorities have NIH grants, (d) fewer women reported success in moving from a mentored to an independent NIH award, and (e) women in the most recent graduation cohort reported spending less time on research than men. Collectively, these results suggest that additional efforts need to be made to recruit women and minorities into MD-PhD programs and, once recruited, to understand the drivers behind the differences that have emerged in their career paths.

MD-PhD programs were established in the 1950s as a new curriculum for training physician-scientists. Since then, the number of programs has grown considerably; however, concerns about the health of the US physician-scientist workforce have grown, as well. The largest attempt to date to assess whether MD-PhD programs are fulfilling their mission was the national MD-PhD program outcomes study, which was released as an American Association of Medical Colleges report in 2018. That study gathered information on 10,591 graduates of 80 MD-PhD programs over 50 years and concluded that most graduates have followed careers consistent with their training. Here, we provide additional analysis, drawing on survey data provided by 64.1% of alumni (75.9% of alumni with valid email addresses), plus program-supplied current workplace data for survey nonresponders to examine the relationships between medical specialty choices, training duration, research effort, and success in obtaining research funding. The results show that residency choices affect critical aspects of the physician-scientist career path, including where graduates work, how long it takes them to obtain an independent appointment in academia, and the amount of their professional time that is devoted to research. Entrants into MD-PhD programs are older, on average, now than when the programs were first established and are taking longer to graduate and complete postgraduate training. Although we found a positive relationship between professional effort devoted to research and the likelihood of having research funding, we found little evidence that the increase in training duration produces an increase in subsequent research effort. These data should provide both guidance for anyone considering this career path and insights for those who train and hire the next generation of physician-scientists.

G protein–coupled receptors are critical mediators of platelet activation whose signaling can be modulated by members of the regulator of G protein signaling (RGS) family. The 2 most abundant RGS proteins in human and mouse platelets are RGS10 and RGS18. While each has been studied individually, critical questions remain about the overall impact of this mode of regulation in platelets. Here, we report that mice missing both proteins show reduced platelet survival and a 40% decrease in platelet count that can be partially reversed with aspirin and a P2Y12 antagonist. Their platelets have increased basal (TREM)-like transcript-1 expression, a leftward shift in the dose/response for a thrombin receptor–activating peptide, an increased maximum response to adenosine 5′-diphosphate and TxA2, and a greatly exaggerated response to penetrating injuries in vivo. Neither of the individual knockouts displays this constellation of findings. RGS10−/− platelets have an enhanced response to agonists in vitro, but platelet count and survival are normal. RGS18−/− mice have a 15% reduction in platelet count that is not affected by antiplatelet agents, nearly normal responses to platelet agonists, and normal platelet survival. Megakaryocyte number and ploidy are normal in all 3 mouse lines, but platelet recovery from severe acute thrombocytopenia is slower in RGS18−/− and RGS10−/−18−/− mice. Collectively, these results show that RGS10 and RGS18 have complementary roles in platelets. Removing both at the same time discloses the extent to which this regulatory mechanism normally controls platelet reactivity in vivo, modulates the hemostatic response to injury, promotes platelet production, and prolongs platelet survival.

Extensive studies have detailed the molecular regulation of individual components of the hemostatic system, including platelets, coagulation factors, and regulatory proteins. Questions remain, however, about how these elements are integrated at the systems level within a rapidly changing physical environment. To answer some of these questions, we developed a puncture injury model in mouse jugular veins that combines high-resolution, multimodal imaging with functional readouts in vivo. The results reveal striking spatial regulation of platelet activation and fibrin formation that could not be inferred from studies performed ex vivo. As in the microcirculation, where previous studies have been performed, gradients of platelet activation are readily apparent, as is an asymmetrical distribution of fibrin deposition and thrombin activity. Both are oriented from the outer to the inner surface of the damaged vessel wall, with a greater extent of platelet activation and fibrin accumulation on the outside than the inside. Further, we show that the importance of P2Y(12) signaling in establishing a competent hemostatic plug is related to the size of the injury, thus limiting its contribution to hemostasis to specific physiologic contexts. Taken together, these studies offer insights into the organization of hemostatic plugs, provide a detailed understanding of the adverse bleeding associated with a widely prescribed class of antiplatelet agents, and highlight differences between hemostasis and thrombosis that may suggest alternative therapeutic approaches.

There is growing concern that the physician-scientist is endangered due to a leaky training pipeline and prolonged time to scientific independence (1). The NIH Physician-Scientist Workforce Working Group has concluded that as many as 1,000 individuals will need to enter the pipeline each year to sustain the workforce (2). Moreover, surveys of postgraduate training programs document considerable variability in disposition and infrastructure (3). Programs can be broadly grouped into two classes: physician-scientist training programs (PSTPs) that span residency and fellowship training, and research-in-residency programs (RiRs), which are limited to residency but trainees are able to match into PSTPs upon transitioning to fellowship (Figure 1). Funding sources for RiRs and PSTPs are varied and include NIH KL2 and T32 awards, charitable foundations, philanthropy, and institutional support. Furthermore, standards for research training and tools for evaluating programmatic success are lacking. Here, we share consensus generated from iterative workshops hosted by the Alliance of Academic Internal Medicine (AAIM) and the student-led American Physician Scientists Association (APSA).

We have previously shown in the microcirculation that gradients of platelet agonists develop during the hemostatic response as a consequence of the solute transport properties within the evolving platelet mass. These agonist gradients result in the formation of a gradient of platelet activation extending from the site of vascular injury. Here we utilized a combined light and electron microscopy approach to examine the organization of hemostatic plugs formed in response to larger injuries that result in significant bleeding in the mouse jugular vein. The results show that the hemostatic response to vessel wall puncture in a large vein is similarly characterized by a gradient of platelet activation. Specifically, we found that 1) platelets are the primary cellular component of hemostatic plugs in large veins; 2) platelet activation is heterogeneous, with fully activated and degranulated platelets present at the interface of the damaged vessel wall and extending to the extravascular side of hemostatic plugs, while platelets aggregated on the luminal side of plugs were minimally activated; 3) fibrin, and by extension thrombin activity, is localized primarily in the extravascular space; 4) P2Y 12 antagonism impairs platelet aggregation, resulting in a prolonged bleeding time when the hole created by the puncture injury does not completely fill with platelets; and 5) the importance of P2Y 12 signaling in establishing a hemostatically competent plug is related to the size of the hole through which blood escapes the vasculature. Taken together, these results provide novel insights into the mechanisms of hemostatic plug formation in vivo, demonstrate in detail the mechanism by which P2Y 12 antagonists result in bleeding, and highlight differences in the composition and morphology of hemostatic plugs versus pathologic thrombi.

Endogenous anticoagulants, including antithrombin III, tissue factor pathway inhibitor, and activated protein C (aPC) provide a biochemical means to inhibit thrombin generation and/or activity. In addition, we have previously shown that platelet mass retraction prevents the escape and exchange of solutes, thereby providing a biophysical mechanism to limit thrombin generation and activity. The relative contribution of each of these biochemical and biophysical mechanisms to the termination of thrombin activity during the hemostatic response in vivo remains poorly understood. Here, using computational simulations coupled with in vivo experimental models, we tested the hypotheses that 1) platelet mass retraction itself is physiologically relevant to inhibit thrombin generation; and 2) that given the spatial localization of protein C activation on the endothelial cell surface, aPC mediated thrombin inhibition has negligible effects on the hemostatic response. Our computational model has several innovative aspects, including 3D representations of the vessel, extravascular space, and injury, blood flow, and a simplified but anatomically correct model of coagulation. Our simulations show that tissue factor localization combined with flow exiting the injury limit thrombin generation/activity to the extravascular space. Our experiments using a mouse jugular puncture injury model show rich deposition of fibrin in the extravascular space and little to no fibrin in the luminal side. Additionally, our simulations illustrate how platelet mass retraction inhibits thrombin generation by decreasing the delivery of substrates, such as prothrombin and factor X, and at the same time it limits thrombin activity by constraining its movement. These effects are mediated by physical forces only and are independent of the biochemical inhibitory pathways. Our in vivo experiments using aPC inhibitory antibodies show that lack of aPC mediated thrombin inhibition has negligible effects on the hemostatic response. In conclusion, this study suggests that platelet mass retraction is an underappreciated mechanism that negatively regulates thrombin generation, while aPC mediated inhibition of thrombin generation plays a minor role in hemostasis.

We are living in a golden age of biomedical research in which it is increasingly feasible to translate fundamental discoveries into new diagnostic and therapeutic approaches to human illnesses. Inherited diseases are being cured with gene therapy. Cancer cells are being eliminated with less toxic small molecule inhibitors and reengineered T-cells. Direct connections are being made between the central nervous system and prosthetic devices. These efforts are being led by scientists and engineers, some of whom are also physicians. This article is intended to help anyone considering a career as a physician–scientist, but unsure about how best to begin. It is also intended for faculty, staff, and parents who are on the front lines of advising talented students about the options that they have for their future. With this in mind, I have tried to answer common questions about MD/PhD programs, but I have also included information about other paths to becoming a physician who does research.

Essentials Platelet packing density in a hemostatic plug limits molecular movement to diffusion. A diffusion-dependent steep thrombin gradient forms radiating outwards from the injury site. Clot retraction affects the steepness of the gradient by increasing platelet packing density. Together, these effects promote hemostatic plug core formation and inhibit unnecessary growth. Summary Background Hemostasis studies performed in vivo have shown that hemostatic plugs formed after penetrating injuries are characterized by a core of highly activated, densely packed platelets near the injury site, covered by a shell of less activated and loosely packed platelets. Thrombin production occurs near the injury site, further activating platelets and starting the process of platelet mass retraction. Tightening of interplatelet gaps may then prevent the escape and exchange of solutes. Objectives To reconstruct the hemostatic plug macro- and micro-architecture and examine how platelet mass contraction regulates solute transport and solute concentration in the gaps between platelets. Methods Our approach consisted of three parts. First, platelet aggregates formed in vitro under flow were analyzed using scanning electron microscopy to extract data on porosity and gap size distribution. Second, a three-dimensional (3-D) model was constructed with features matching the platelet aggregates formed in vitro. Finally, the 3-D model was integrated with volume and morphology measurements of hemostatic plugs formed in vivo to determine how solutes move within the platelet plug microenvironment. Results The results show that the hemostatic mass is characterized by extremely narrow gaps, porosity values even smaller than previously estimated and stagnant plasma velocity. Importantly, the concentration of a chemical species released within the platelet mass increases as the gaps between platelets shrink. Conclusions Platelet mass retraction provides a physical mechanism to establish steep chemical concentration gradients that determine the extent of platelet activation and account for the core-and-shell architecture observed in vivo.

Most platelet agonists activate platelets by binding to G-protein-coupled receptors. We have shown previously that a critical node in the G-protein signaling network in platelets is formed by a scaffold protein, spinophilin (SPL), the tyrosine phosphatase, Src homology region 2 domain-containing phosphatase-1 (SHP-1), and the regulator of G-protein signaling family member, RGS18. Here, we asked whether SPL and other RGS18 binding proteins such as 14-3-3γ regulate platelet reactivity by sequestering RGS18 and, if so, how this is accomplished. The results show that, in resting platelets, free RGS18 levels are relatively low, increasing when platelets are activated by thrombin. Free RGS18 levels also rise when platelets are rendered resistant to activation by exposure to prostaglandin I2 (PGI2) or forskolin, both of which increase platelet cyclic adenosine monophosphate (cAMP) levels. However, the mechanism for raising free RGS18 is different in these 2 settings. Whereas thrombin activates SHP-1 and causes dephosphorylation of SPL tyrosine residues, PGI2 and forskolin cause phosphorylation of SPL Ser94 without reducing tyrosine phosphorylation. Substituting alanine for Ser94 blocks cAMP-induced dissociation of the SPL/RGS/SHP-1 complex. Replacing Ser94 with aspartate prevents formation of the complex and produces a loss-of-function phenotype when expressed in mouse platelets. Together with the defect in platelet function we previously observed in SPL(-/-) mice, these data show that (1) regulated sequestration and release of RGS18 by intracellular binding proteins provides a mechanism for coordinating activating and inhibitory signaling networks in platelets, and (2) differential phosphorylation of SPL tyrosine and serine residues provides a key to understanding both.

The local microenvironment within an evolving hemostatic plug shapes the distribution of soluble platelet agonists, resulting in a gradient of platelet activation. We previously showed that thrombin activity at a site of vascular injury is spatially restricted, resulting in robust activation of a subpopulation of platelets in the hemostatic plug core. In contrast, adenosine 5'-diphosphate (ADP)/P2Y12 signaling contributes to the accumulation of partially activated, loosely packed platelets in a shell overlying the core. The contribution of the additional platelet agonists thromboxane A2 (TxA2) and epinephrine to this hierarchical organization was not previously shown. Using a combination of genetic and pharmacologic approaches coupled with real-time intravital imaging, we show that TxA2 signaling is critical and nonredundant with ADP/P2Y12 for platelet accumulation in the shell region but not required for full platelet activation in the hemostatic plug core, where thrombin activity is highest. In contrast, epinephrine signaling is dispensable even in the presence of a P2Y12 antagonist. Finally, dual P2Y12 and thrombin inhibition does not substantially inhibit hemostatic plug core formation any more than thrombin inhibition alone, providing further evidence that thrombin is the primary driver of platelet activation in this region. Taken together, these studies show for the first time how thrombin, P2Y12, and TxA2 signaling are coordinated during development of a hierarchical organization of hemostatic plugs in vivo and provide novel insights into the impact of dual antiplatelet therapy on hemostasis and thrombosis.

Blood coagulation involves activation of platelets and coagulation factors. At the interface of these two processes resides the lipid phosphatidylserine. Activated platelets expose phosphatidylserine on their outer membrane leaflet and activated clotting factors assemble into enzymatically active complexes on the exposed lipid, ultimately leading to the formation of fibrin. Here, we describe how small peptide and peptidomimetic probes derived from the lipid binding domain of the protein myristoylated alanine-rich C-kinase substrate (MARCKS) bind to phosphatidylserine exposed on activated platelets and thereby inhibit fibrin formation. The MARCKS peptides antagonize the binding of factor Xa to phosphatidylserine and inhibit the enzymatic activity of prothrombinase. In whole blood under flow, the MARCKS peptides colocalize with, and inhibit fibrin cross-linking, of adherent platelets. In vivo, we find that the MARCKS peptides circulate to remote injuries and bind to activated platelets in the inner core of developing thrombi.

Platelets express ≥2 members of the regulators of G protein signaling (RGS) family. Here, we have focused on the most abundant, RGS10, examining its impact on the hemostatic response in vivo and the mechanisms involved. We have previously shown that the hemostatic thrombi formed in response to penetrating injuries consist of a core of fully activated densely packed platelets overlaid by a shell of less-activated platelets responding to adenosine 5'-diphosphate (ADP) and thromboxane A

The local microenvironment within an evolving hemostatic plug shapes the distribution of soluble platelet agonists, resulting in a gradient of platelet activation emanating from the site of injury. While thrombin and ADP mediated platelet activation are clearly critical for establishment of this gradient, it remains unclear to what extent there is overlap of these and other platelet signaling pathways in time and space. In other words, does a single agonist dominate the platelet activation state within specific regions of a hemostatic plug, or do multiple agonist pathways need to be integrated to produce the gradient of platelet activation observed? Further, how do the relationships among different agonists change in the face of anti-platelet or anti-coagulant therapy and what are the consequences for hemostatic plug organization? Here, we used a combination of genetic and pharmacologic approaches coupled with real-time intravital imaging to examine how thrombin, thromboxane A 2 , P2Y 12 and epinephrine signaling are coordinated in time and space to regulate the development of platelet activation gradients at a site of vascular injury in vivo. We found that both thromboxane A 2 /TP signaling and ADP/P2Y 12 signaling are required for accumulation of minimally activated platelets in the outer shell region of hemostatic plugs. Interestingly, dual inhibition of both thromboxane and P2Y 12 signaling did not have an additive effect, but rather was similar to inhibition of either pathway alone. Epinephrine, which activates a Gi signaling pathway similar to ADP/P2Y 12 , was completely dispensable for hemostatic plug formation, even in the absence of P2Y 12 signaling. Finally, inhibition of P2Y 12 in the setting of sub-maximal thrombin activity revealed no role for P2Y 12 signaling in the development of robust platelet activation within the hemostatic plug core region. Taken together, these data shed new light on the way multiple platelet signaling pathways are integrated during the hemostatic response in vivo, and predict the outcome of therapeutically targeting specific platelet signaling pathways alone and in combination on hemostatic plug organization.

Previous studies using intravital microscopy have shown that hemostatic plugs formed in the mouse microvasculature have a characteristic architecture in which the extent of platelet activation reflects gradients in the distribution of platelet agonists radiating outwards from the injury site. In that setting, we found minimal overlap of thrombin and ADP signaling, with thrombin primarily responsible for robust platelet activation close to the injury site and P2Y 12 -mediated ADP signaling resulting in accumulation of minimally activated platelets. Here we have taken these studies a major step forward by integrating fluorescence with scanning electron microscopy. Hemostatic plugs produced by needle injury in mouse jugular veins were imaged in situ 1 to 20 min after injury. The results show with unprecedented detail what could only be inferred previously, showing that platelet size, morphology and packing density vary remarkably depending on spatial localization within the hemostatic plug. The intraluminal and extravascular portions of the hemostatic mass presented distinct architectures. A large mass comprised almost exclusively of platelets was observed on the interior surface of the vein. Platelets closest to the injury edge had a highly activated morphology, including P-selectin surface expression, dense packing and platelet fragmentation, while those farther from the injury edge often remained discoid. In contrast, the extravascular portion of the hemostatic mass was rich in densely-packed, platelet-derived fragments intertwined with fibrin. Hemostatic plugs from mice treated with a P2Y 12 inhibitor were significantly smaller. The platelet activation gradient described above was less apparent and, notably, fragmentation of the platelets close to the injury edge was not observed with the inhibitor present. In conclusion, our findings indicate that 1) the development of a platelet activation gradient is a conserved feature of the hemostatic response across different vessels, 2) fragmentation of platelets closest to the injury site occurs very rapidly following injury, and 3) clinically relevant platelet signaling pathways play a role in regulating its formation.

Hemostatic thrombi formed after a penetrating injury have a distinctive structure in which a core of highly activated, closely packed platelets is covered by a shell of less-activated, loosely packed platelets. We have shown that differences in intrathrombus molecular transport emerge in parallel with regional differences in platelet packing density and predicted that these differences affect thrombus growth and stability. Here we test that prediction in a mouse vascular injury model. The studies use a novel method for measuring thrombus contraction in vivo and a previously characterized mouse line with a defect in integrin αIIbβ3 outside-in signaling that affects clot retraction ex vivo. The results show that the mutant mice have a defect in thrombus consolidation following vascular injury, resulting in an increase in intrathrombus transport rates and, as predicted by computational modeling, a decrease in thrombin activity and platelet activation in the thrombus core. Collectively, these data (1) demonstrate that in addition to the activation state of individual platelets, the physical properties of the accumulated mass of adherent platelets is critical in determining intrathrombus agonist distribution and platelet activation and (2) define a novel role for integrin signaling in the regulation of intrathrombus transport rates and localization of thrombin activity.

Hemostatic thrombi formed after a penetrating injury have a heterogeneous architecture in which a core of highly activated, densely packed platelets is covered by a shell of less-activated, loosely packed platelets. In the first manuscript in this series, we show that regional differences in intrathrombus protein transport rates emerge early in the hemostatic response and are preserved as the thrombus develops. Here, we use a theoretical approach to investigate this process and its impact on agonist distribution. The results suggest that hindered diffusion, rather than convection, is the dominant mechanism responsible for molecular movement within the thrombus. The analysis also suggests that the thrombus core, as compared with the shell, provides an environment for retaining soluble agonists such as thrombin, affecting the extent of platelet activation by establishing agonist-specific concentration gradients radiating from the site of injury. This analysis accounts for the observed weaker activation and relative instability of platelets in the shell and predicts that a failure to form a tightly packed thrombus core will limit thrombin accumulation, a prediction tested by analysis of data from mice with a defect in clot retraction.

Purpose of review Several decades of work by many investigators have elucidated the major signaling pathways responsible for platelet activation. Still to be fully understood is how these pathways are integrated into a single network and how changing conditions within a growing thrombus affect that network. In this review we will consider some of the recent studies that address these issues and describe a model that provides insights into platelet activation as it occurs in vivo. Recent findings Genetic and pharmacologic studies performed in vivo have demonstrated that platelet activation during hemostasis and thrombosis is heterogeneous. Those studies indicate that distinct platelet activation pathways are not merely redundant, but are coordinated in time and space to achieve an optimal response. This coordination is achieved at least in part by the evolving distribution of platelet agonists and changes in solute transport within a hemostatic plug. Summary Studies examining the coordination of platelet signaling in time and space continue to increase our understanding of hemostasis and thrombosis. In addition to helping to decipher platelet biology, the results have implications for the understanding of new and existing antiplatelet agents and their potential risks.

Introduction: Platelet recruitment to generate a thrombus is key to bleeding cessation. That recruitment is dependent on a series of platelet activation processes that include adhesion to the exposed vessel matrix, platelet-platelet adhesion and platelet granule release. How platelet activation is patterned to generate a thrombus has previously been studied by intravital light microscopy, two-photon microscopy and scanning electron microscopy at resolutions insufficient to infer platelet activation at the level of the individual platelet. Here, we present a collaborative effort to stratify spatially the extent of platelet activation at the cellular level in a mouse jugular vein puncture model. We used wide-area transmission electron microscopy (WA-TEM) and serial block face scanning electron microscopy (SBF-SEM) at a resolution of 3 to 100 nm across whole thrombi to determine activation state of individual platelets. Our results, indicate a pattern of platelet stratification within the puncture wound that varies in a time-dependent manner with distinct structural stages in the formation of the thrombus. Methods: Jugular vein thrombi from C57BL/6 mice were collected 1, 5, or 20 min after a 300 µm needle puncture and prepared for EM imaging. For WA-TEM, hundreds of overlapping 3 nm resolution images were acquired using a gondimeter stage. The images sets were aligned using NIH Fiji software to create a single high-res, thrombus-wide image. Individual platelets were stratified into morphologically defined activation states (1: no activation, 2: decreased granule number, 3: no visible granules left, 4: hollow inside). The spatial distribution of platelet stratification was analyzed using iVision software. For SBF-SEM, 100-nm XY-resolution SEM images were collected every 200 nm and 20 nm XY-resolution images every 20 µm. Semi-automated stratification of platelet activation state in individual slices of the thrombus were combined into a 3-D representation using Amira software. Volumetric distributions of platelets with respect to the puncture hole and the vascular wall were quantified. Results: Thrombus Formation Stage 1 (anchor and extend) -- One min post puncture, platelets were anchored in clumps along the exposed vessel wall. Near the damaged vessel wall was a peripheral layer of activated or degranulated platelets (states 3 and 4) covered by additional layers of less-activated platelets (state 1 and 2). Short cylindrical ingrowths extended into the 300 µm hole. Unexpectedly, large aggregates of platelets with a mixture of activation states (states 1 - 4) were found extending from these anchor points into the hole and vertically into the intravascular space. Aggregate surface layers were composed mostly of degranulated platelets (states 3 and 4). Less than 40% of neighboring platelets were of the same activation state as their neighbor. Surprisingly, Stage 2 (cap and erect) -- At 5 min after injury, the puncture hole was capped. ~70% of platelets neighboring degranulated platelets (3 and 4) formed visible aggregates within the thrombus. These aggregates were found along the exposed vessel wall and encasing vertical platelet aggregate towers containing a mixture of platelets in different states (1 - 4). Towers were typically separated by large cavities. SBF-SEM images, a machine-based, unbiased sampling of the underlying platelet distribution, revealed that ~10% of the platelet volume in the puncture hole of the thrombus and the intravascular towers contained largely degranulated platelets, similar to the data from WA-TEM. Stage 3 (infill and remodeling) - At 20 min post-puncture, the thrombus was filled with a mixture of platelets of varying activation states, which surrounded central, vertical aggregates (towers) of degranulated platelets seen at 5 min. Only minor cavity space was apparent. The intravascular surface of the thrombus was covered with an ~10 platelet-thick layer of loosely packed, variably activated platelets (states 1 - 4). Conclusions: Our results demonstrate dynamic spatial patterns of platelet activation within a forming puncture-wound thrombus. Such patterns yield insights into thrombus formation and suggest the need to reference platelets defects and anti-thrombotics drugs against new models. Figure Disclosures No relevant conflicts of interest to declare.

[This corrects the article DOI: 10.1371/journal.pone.0064265.].

Semaphorins are cell surface proteins sharing an extracellular Sema motif that binds to plexin family receptors, among others [1]. We have shown previously that Sema4D is expressed by platelets, serving as a contact-dependent amplifier of platelet activation by promoting activation of the tyrosine kinase, Syk, downstream of the collagen receptor, GP VI/FcRγ [2–4]. Deletion of Sema4D impairs thrombus growth in mice by reducing the number of fully-activated, stably-adherent platelets in the region closest to the vessel wall [5]. Sema4D(−/−) platelets have diminished responses to collagen, but normal responses to thrombin, TxA2 and ADP [2, 4]. Collagen-induced Syk activation and the subsequent activation of phospholipase Cγ2 are most robust when Sema4D is present and platelets are allowed to form stable contacts [4]. These observations have helped to define the role of platelet Sema4D. In the setting of vascular injury, Syk is activated by binding to a phosphorylated immunoreceptor tyrosine-based activation motif (ITAM) in FcRγ. However, while our previous studies show that Sema4D does not amplify G protein-dependent signaling, left unsettled was whether signal amplification is specific for GPVI or applies to other ITAM receptors as well. Here we have addressed that issue. Human platelets express two other ITAM-containing receptors, Clec-2 and FcR-IIA. Mouse platelets express Clec-2 [6]. Unlike GPVI/FcRγ and FcR-IIA, Clec-2 contains only half of an ITAM motif or “hemi-ITAM” (YXXL). Signaling occurs when two molecules of phosphorylated Clec-2 engage a single molecule of Syk [6, 7]. The known Clec-2 ligands are the snake venom toxin, rhodocytin [6] and podoplanin. Podoplanin/Clec-2 interactions play an essential role in separating the lymphatic and vascular systems during embryonic development [8–10].

Semaphorin 4D (Sema4D) is a transmembrane protein that supports contact-dependent amplification of platelet activation by collagen before being gradually cleaved by the metalloprotease ADAM17, as we have previously shown. Cleavage releases a soluble 120-kDa exodomain fragment for which receptors exist on platelets and endothelial cells. Here we have examined the mechanism that regulates Sema4D exodomain cleavage. The results show that the membrane-proximal cytoplasmic domain of Sema4D contains a binding site for calmodulin within the polybasic region Arg762-Lys779. Coprecipitation studies show that Sema4D and calmodulin are associated in resting platelets, forming a complex that dissociates upon platelet activation by the agonists that trigger Sema4D cleavage. Inhibiting calmodulin with W7 or introducing a membrane-permeable peptide corresponding to the calmodulin-binding site is sufficient to trigger the dissociation of Sema4D from calmodulin and initiate cleavage. Conversely, deletion of the calmodulin-binding site causes constitutive shedding of Sema4D. These results show that (1) Sema4D is a calmodulin-binding protein with a site of interaction in its membrane-proximal cytoplasmic domain, (2) platelet agonists cause dissociation of the calmodulin-Sema4D complex, and (3) dissociation of the complex is sufficient to trigger ADAM17-dependent cleavage of Sema4D, releasing a bioactive fragment.

Achieving hemostasis following vascular injury requires the rapid accumulation of platelets and fibrin. Here we used a combination of confocal intravital imaging, genetically engineered mice, and antiplatelet agents to determine how variations in the extent of platelet activation following vascular injury arise from the integration of different elements of the platelet-signaling network. Two forms of penetrating injury were used to evoke the hemostatic response. Both produced a hierarchically organized structure in which a core of fully activated platelets was overlaid with an unstable shell of less-activated platelets. This structure emerged as hemostasis was achieved and persisted for at least 60 minutes following injury, its organization at least partly reflecting agonist concentration gradients. Thrombin activity and fibrin formation were found primarily in the innermost core. As proposed previously, greater packing density in the core facilitated contact-dependent signaling and limited entry of plasma-borne molecules visualized with fluorophores coupled to dextran and albumin. Blocking contact-dependent signaling or inhibiting thrombin reduced the size of the core, while the shell was heavily influenced by adenosine 5'-diphosphate and regulators of Gi2-mediated signaling. Thus, the hemostatic response is shown to produce a hierarchical structure arising, in part, from distinct elements of the platelet-signaling network.

We have previously shown that Sema4D expressed on the platelet plasma membrane can be cleaved by the metalloprotease ADAM17, producing a 120-kDa exodomain fragment that retains biological activity and remnant fragments of 24-28 kDa that remain associated with the platelet membrane. This process is modulated by calmodulin. Here we investigated the potential role of protein kinase A (PKA) in these events. Using a pharmacological approach, we now show that inhibition of PKA by H89 is sufficient to induce Sema4D exodomain shedding, while activation of PKA inhibits agonist-initiated shedding. Studies on the regulatory mechanism show that the shedding induced by PKA inhibition is mediated by ADAM17, but, unlike agonist-induced shedding, does not involve the dissociation of calmodulin from the Sema4D cytoplasmic domain. In attempt to identify the cleavage sites for shedding, we found that ADAM17 mediates variable cleavages in the juxtamembrane region. Therefore, our data reveal a potential regulatory mechanism for the shedding of Sema4D in platelets.

Summary. Background: Thrombin undergoes convective and diffusive transport, making it difficult to visualize during thrombosis. We developed the first sensor capable of revealing inner clot thrombin dynamics. Methods and results: An Nterminal-azido thrombin-sensitive fluorescent peptide (ThS-P) with a thrombin-releasable quencher was linked to anti-CD41 using click chemistry to generate a thrombin-sensitive platelet binding sensor (ThS-Ab). Rapid thrombin cleavage of ThS-P (Km = 40.3 lM, kcat =1 .5 s )1 ) allowed thrombin monitoring by ThS-P or ThS-Ab in blood treated with 2–25 pM tissue factor (TF). Individual platelets had > 20-fold more ThS-Ab fluorescence after clotting. In a microfluidic assay of whole blood perfusion over collagen ± linked TF (wall shear rate = 100 s )1 ), ThS-Ab fluorescence increased between 90 and 450 s for 0.1–1 molecule-TF lm )2 and co-localized with platelets near fibrin. Without TF, neither thrombin nor fibrin was detected on the platelet deposits by 450 s. Using a microfluidic device to control the pressure drop across a thrombus forming on a porous collagen/TF plug (521 s )1 ), thrombin and fibrin were detected at the thrombus–collagen interface at a zero pressure drop, whereas 80% less thrombin was detected at 3200 Pa in concert with fibrin polymerizing within the collagen. With anti-mouse CD41 ThS-Ab deployed in a mouse laser injury model, the highest levels of thrombin arose between 40 and 160 s nearest the injury site where fibrin co-localized and where the thrombus was most mechanically stable. Conclusion: ThS-Ab reveals thrombin locality, which depends on surface TF, flow and intrathrombus pressure gradients.

Platelets are essential for normal hemostasis, but close regulation is required to avoid the destructive effects of either inappropriate platelet activation or excessive responses to injury. Here, we describe a novel complex comprising the scaffold protein, spinophilin (SPL), and the tyrosine phosphatase, SHP-1, and show that it can modulate platelet activation by sequestering RGS10 and RGS18, 2 members of the regulator of G protein signaling family. We also show that SPL/RGS/SHP1 complexes are present in resting platelets where constitutive phosphorylation of SPL(Y398) creates an atypical binding site for SHP-1. Activation of the SHP-1 occurs on agonist-induced phosphorylation of SHP-1(Y536), triggering dephosphorylation and decay of the SPL/RGS/SHP1 complex. Preventing SHP-1 activation blocks decay of the complex and produces a gain of function. Conversely, deleting spinophilin in mice inhibits platelet activation. It also attenuates the rise in platelet cAMP normally caused by endothelial prostacyclin (PGI2). Thus, we propose that the role of the SPL/RGS/SHP1 complex in platelets is time and context dependent. Before injury, the complex helps maintain the quiescence of circulating platelets by maximizing the impact of PGI2. After injury, the complex gradually releases RGS proteins, limiting platelet activation and providing a mechanism for temporal coordination of pro thrombotic and antithrombotic inputs.