The National MD-PhD Program Outcomes Study: career paths followed by Black and Hispanic graduates.

Previous studies on attrition from MD-PhD programs have shown that students who self-identify as Black are more likely to withdraw before graduating than Hispanic students and students not from groups underrepresented in medicine (non-UIM). Here, we analyzed data collected for the National MD-PhD Program Outcomes Study, a national effort to track the careers of over 10,000 individuals who have graduated from MD-PhD programs over the past 60 years. On average, Black trainees took slightly longer to graduate, were less likely to choose careers in academia, and were more likely to enter nonacademic clinical practice; although, none of these differences were large. Black graduates were also more likely to choose careers in surgery or internal medicine, or entirely forego residency, and less likely to choose pediatrics, pathology, or neurology. Among those in academia, average research effort rates self-reported by Black, Hispanic, and non-UIM alumni were indistinguishable, as were rates of obtaining research grants and mentored training awards. However, the proportion of Black and Hispanic alumni who reported having NIH research grants was lower than that of non-UIM alumni, and the NIH career development to research project grant (K-to-R) conversion rate was lower for Black alumni. We propose that the reasons for these differences reflect experiences before, during, and after training and, therefore, conclude with action items that address each of these stages.

Megakaryocyte-induced contraction of plasma clots: cellular mechanisms and structural mechanobiology.

ABSTRACT: Nonmuscle cell contractility is an essential feature underlying diverse cellular processes such as motility, morphogenesis, division and genome replication, intracellular transport, and secretion. Blood clot contraction is a well-studied process driven by contracting platelets. Megakaryocytes (MKs), which are the precursors to platelets, can be found in bone marrow and lungs. Although they express many of the same proteins and structures found in platelets, little is known about their ability to engage with extracellular proteins such as fibrin and contract. Here, we have measured the ability of MKs to compress plasma clots. Megakaryocytes derived from human induced pluripotent stem cells (iPSCs) were suspended in human platelet-free blood plasma and stimulated with thrombin. Using real-time macroscale optical tracking, confocal microscopy, and biomechanical measurements, we found that activated iPSC-derived MKs (iMKs) caused macroscopic volumetric clot shrinkage, as well as densification and stiffening of the fibrin network via fibrin-attached plasma membrane protrusions undergoing extension-retraction cycles that cause shortening and bending of fibrin fibers. Contraction induced by iMKs involved 2 kinetic phases with distinct rates and durations. It was suppressed by inhibitors of nonmuscle myosin IIA, actin polymerization, and integrin αIIbß3-fibrin interactions, indicating that the molecular mechanisms of iMK contractility were similar or identical to those in activated platelets. Our findings provide new insights into MK biomechanics and suggest that iMKs can be used as a model system to study platelet contractility. Physiologically, the ability of MKs to contract plasma clots may play a role in the mechanical remodeling of intravascular blood clots and thrombi.

A regulatory node involving Gαq, PLCß, and RGS proteins modulates platelet reactivity to critical agonists.

BACKGROUND: Most platelet agonists work through G protein-coupled receptors, activating pathways that involve members of the Gq, Gi, and G12/G13 families of heterotrimeric G proteins. Gq signaling has been shown to be critical for efficient platelet activation. Growing evidence suggests that regulatory mechanisms converge on G protein-coupled receptors and Gq to prevent overly robust platelet reactivity. OBJECTIVES: To identify and characterize mechanisms by which Gq signaling is regulated in platelets. METHODS: Based on our prior experience with a Gαi2 variant that escapes regulation by regulator of G protein signaling (RGS) proteins, a Gαq variant was designed with glycine 188 replaced with serine (G188S) and then incorporated into a mouse line so that its effects on platelet activation and thrombus formation could be studied in vitro and in vivo. RESULTS AND CONCLUSIONS: As predicted, the G188S substitution in Gαq disrupted its interaction with RGS18. Unexpectedly, it also uncoupled PLCß-3 from activation by platelet agonists as evidenced by a loss rather than a gain of platelet function in vitro and in vivo. Binding studies showed that in addition to preventing the binding of RGS18 to Gαq, the G188S substitution also prevented the binding of PLCß-3 to Gαq. Structural analysis revealed that G188 resides in the region that is also important for Gαq binding to PLCß-3 in platelets. We conclude that the Gαq signaling node is more complex than that has been previously understood, suggesting that there is cross-talk between RGS proteins and PLCß-3 in the context of Gαq signaling.

Gaps between college and starting an MD-PhD program are adding years to physician-scientist training time.

The average age when physician-scientists begin their career has been rising. Here, we focused on one contributor to this change: the increasingly common decision by candidates to postpone applying to MD-PhD programs until after college. This creates a time gap between college and medical school. Data were obtained from 3544 trainees in 73 programs, 72 program directors, and AAMC databases. From 2013 to 2020, the prevalence of gaps rose from 53% to 75%, with the time usually spent doing research. Gap prevalence for MD students also increased but not to the same extent and for different reasons. Differences by gender, underrepresented status, and program size were minimal. Most candidates who took a gap did so because they believed it would improve their chances of admission, but gaps were as common among those not accepted to MD-PhD programs as among those who were. Many program directors preferred candidates with gaps, believing without evidence that gaps reflects greater commitment. Although candidates with gaps were more likely to have a publication at the time of admission, gaps were not associated with a shorter time to degree nor have they been shown to improve outcomes. Together, these observations raise concerns that, by promoting gaps after college, current admissions practices have had unintended consequences without commensurate advantages.

Pearls of wisdom for aspiring physician-scientist residency applicants and program directors.

Postgraduate physician-scientist training programs (PSTPs) enhance the experiences of physician-scientist trainees following medical school graduation. PSTPs usually span residency and fellowship training, but this varies widely by institution. Applicant competitiveness for these programs would be enhanced, and unnecessary trainee anxiety relieved, by a clear understanding of what factors define a successful PSTP matriculant. Such information would also be invaluable to PSTP directors and would allow benchmarking of their admissions processes with peer programs. We conducted a survey of PSTP directors across the US to understand the importance they placed on components of PSTP applications. Of 41 survey respondents, most were from internal medicine and pediatrics residency programs. Of all components in the application, two elements were considered very important by a majority of PSTP directors: (a) having one or more first-author publications and (b) the thesis advisor's letter. Less weight was consistently placed on factors often considered more relevant for non-physician-scientist postgraduate applicants - such as US Medical Licensing Examination scores, awards, and leadership activities. The data presented here highlight important metrics for PSTP applicants and directors and suggest that indicators of scientific productivity and commitment to research outweigh traditional quantitative measures of medical school performance.

Thrombin spatial distribution determines protein C activation during hemostasis and thrombosis.

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.

The contribution of TFPIα to the hemostatic response to injury in mice.

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 (TfpiΔα/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: TfpiΔα/Δα mice were viable and fertile. Plasma TFPI levels were normal in the TfpiΔα/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 TfpiΔα/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.

A Human Vascular Injury-on-a-Chip Model of Hemostasis.

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. This microphysiological model replicates the living endothelium, multilayered microarchitecture, and procoagulant activity of human blood vessels, and is also equipped with a microneedle that is 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.

Core and shell platelets of a thrombus: A new microfluidic assay to study mechanics and biochemistry.

BACKGROUND: Hemostatic clots have a P-selectin positive platelet core covered with a shell of P-selectin negative platelets. OBJECTIVE: To develop a new human blood microfluidic assay to interrogate core/shell mechanics. METHODS: A 2-stage assay perfused whole blood over collagen/± tissue factor (TF) for 180 seconds at 100 s-1 wall shear rate, followed by buffer perfusion at either 100 s-1 (venous) or 1000 s-1 (arterial). This microfluidic assay used an extended channel height (120 µm), allowing buffer perfusion well before occlusion. RESULTS: Clot growth on collagen stopped immediately with buffer exchange, revealing ~10% reduction in platelet fluorescence intensity (at 100 s-1) and ~30% (at 1000 s-1) by 1200 seconds. Thrombin generation (on collagen/TF) reduced erosion at either buffer flow rate. P-selectin-positive platelets were stable (no erosion) against 1000 s-1, in contrast to P-selectin negative platelets. Thrombin inhibition (with D-Phe-Pro-Arg-CMK) reduced the number of P-selectin-positive platelets and lowered thrombus stability through the reduction of P-selectin-positive platelets. Interestingly, fibrin inhibition (with H-Gly-Pro-Arg-Pro-OH acetate salt) increased the number of P-selectin-positive platelets but did not lower stability, suggesting that fibrin was only in the core region. Thromboxane inhibition reduced P-selectin-positive platelets and caused a nearly 60% reduction of the clot at arterial buffer flow. P2Y1 antagonism reduced clot size and the number of P-selectin-positive platelets and reduced the stability of P-selectin-negative platelets. CONCLUSION: The 2-stage assay (extended channel height plus buffer exchange) interrogated platelet stability using human blood. Under all conditions, P-selectin-positive platelets never left the clot.

Hemostasis vs. homeostasis: Platelets are essential for preserving vascular barrier function in the absence of injury or inflammation.

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.

RGS10 and RGS18 differentially limit platelet activation, promote platelet production, and prolong platelet survival.

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.

MD-PhD graduates remain underrepresented in orthopaedic surgery: National MD-PhD Program Outcome Survey update.

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.

GRK6 regulates the hemostatic response to injury through its rate-limiting effects on GPCR signaling in platelets.

G protein-coupled receptors (GPCRs) mediate the majority of platelet activation in response to agonists. However, questions remain regarding the mechanisms that provide negative feedback toward activated GPCRs to limit platelet activation and thrombus formation. Here we provide the first evidence that GPCR kinase 6 (GRK6) serves this role in platelets, using GRK6-/- mice generated by CRISPR-Cas9 genome editing to examine the consequences of GRK6 knockout on GPCR-dependent signaling. Hemostatic thrombi formed in GRK6-/- mice are larger than in wild-type (WT) controls during the early stages of thrombus formation, with a rapid increase in platelet accumulation at the site of injury. GRK6-/- platelets have increased platelet activation, but in an agonist-selective manner. Responses to PAR4 agonist or adenosine 5'-diphosphate stimulation in GRK6-/- platelets are increased compared with WT littermates, whereas the response to thromboxane A2 (TxA2) is normal. Underlying these changes in GRK6-/- platelets is an increase in Ca2+ mobilization, Akt activation, and granule secretion. Furthermore, deletion of GRK6 in human MEG-01 cells causes an increase in Ca2+ response and PAR1 surface expression in response to thrombin. Finally, we show that human platelet activation in response to thrombin causes an increase in binding of GRK6 to PAR1, as well as an increase in the phosphorylation of PAR1. Deletion of GRK6 in MEG-01 cells causes a decrease in PAR1 phosphorylation. Taken together, these data show that GRK6 regulates the hemostatic response to injury through PAR- and P2Y12-mediated effects, helping to limit the rate of platelet activation during thrombus growth and prevent inappropriate platelet activation.

The national MD-PhD program outcomes study: Outcomes variation by sex, race, and ethnicity.

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.

The national MD-PhD program outcomes study: Relationships between medical specialty, training duration, research effort, and 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.

Correction: Increased Levels of Plasma Soluble Sema4D in Patients with Heart Failure.

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

Interrelationships between structure and function during the hemostatic response to injury.

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 P2Y12 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.

Training the physician-scientist: views from program directors and aspiring young investigators.

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).