Reduced platelet forces underlie impaired hemostasis in mouse models of MYH9-related disease.

MYH9-related disease patients with mutations in the contractile protein nonmuscle myosin heavy chain IIA display, among others, macrothrombocytopenia and a mild-to-moderate bleeding tendency. In this study, we used three mouse lines, each with one point mutation in the Myh9 gene at positions 702, 1424, or 1841, to investigate mechanisms underlying the increased bleeding risk. Agonist-induced activation of Myh9 mutant platelets was comparable to controls. However, myosin light chain phosphorylation after activation was reduced in mutant platelets, which displayed altered biophysical characteristics and generated lower adhesion, interaction, and traction forces. Treatment with tranexamic acid restored clot retraction in the presence of tPA and reduced bleeding. We verified our findings from the mutant mice with platelets from patients with the respective mutation. These data suggest that reduced platelet forces lead to an increased bleeding tendency in patients with MYH9-related disease, and treatment with tranexamic acid can improve the hemostatic function.

Cytoskeleton Dependent Mobility Dynamics of FcγRIIA Facilitates Platelet Haptotaxis and Capture of Opsonized Bacteria.

Platelet adhesion and spreading at the sites of vascular injury is vital to hemostasis. As an integral part of the innate immune system, platelets interact with opsonized bacterial pathogens through FcγRIIA and contribute to host defense. As mechanoscavangers, platelets actively migrate and capture bacteria via cytoskeleton-rich, dynamic structures, such as filopodia and lamellipodia. However, the role of human platelet FcγRIIA in cytoskeleton-dependent interaction with opsonized bacteria is not well understood. To decipher this, we used a reductionist approach with well-defined micropatterns functionalized with immunoglobulins mimicking immune complexes at planar interfaces and bacteriamimetic microbeads. By specifically blocking of FcγRIIA and selective disruption of the platelet cytoskeleton, we show that both functional FcγRIIA and cytoskeleton are necessary for human platelet adhesion and haptotaxis. The direct link between FcγRIIA and the cytoskeleton is further explored by single-particle tracking. We then demonstrate the relevance of cytoskeleton-dependent differential mobilities of FcγRIIA on bacteria opsonized with the chemokine platelet factor 4 (PF4) and patient-derived anti-PF4/polyanion IgG. Our data suggest that efficient capture of opsonized bacteria during host-defense is governed by mobility dynamics of FcγRIIA on filopodia and lamellipodia, and the cytoskeleton plays an essential role in platelet morphodynamics at biological interfaces that display immune complexes.

Ex vivo anticoagulants affect human blood platelet biomechanics with implications for high-throughput functional mechanophenotyping.

Inherited platelet disorders affecting the human platelet cytoskeleton result in increased bleeding risk. However, deciphering their impact on cytoskeleton-dependent intrinsic biomechanics of platelets remains challenging and represents an unmet need from a diagnostic and prognostic perspective. It is currently unclear whether ex vivo anticoagulants used during collection of peripheral blood impact the mechanophenotype of cellular components of blood. Using unbiased, high-throughput functional mechanophenotyping of single human platelets by real-time deformability cytometry, we found that ex vivo anticoagulants are a critical pre-analytical variable that differentially influences platelet deformation, their size, and functional response to agonists by altering the cytoskeleton. We applied our findings to characterize the functional mechanophenotype of platelets from a patient with Myosin Heavy Chain 9 (MYH9) related macrothrombocytopenia. Our data suggest that platelets from MYH9 p.E1841K mutation in humans affecting platelet non-muscle myosin heavy chain IIa (NMMHC-IIA) are biomechanically less deformable in comparison to platelets from healthy individuals.

Role of Platelet Cytoskeleton in Platelet Biomechanics: Current and Emerging Methodologies and Their Potential Relevance for the Investigation of Inherited Platelet Disorders.

Cytoskeleton is composed of more than 100 proteins and represents a dynamic network of the cellular cytoplasm. Cytoskeletal functions include spatial organization of cellular components, structural connection of the cell with external environment, and biomechanical force generation. Cytoskeleton takes part, at different levels, in all phases of platelet biogenesis: megakaryocyte (MK) differentiation, MK maturation, and platelet formation. In addition, it also plays a major role in each stage of platelet function. Inherited platelet disorders (IPDs) are a group of rare diseases featured by low platelet count and/or impaired platelet function. Over the past decade, the investigation of platelet biomechanics has become a major and highly relevant theme of research due to its implications at every stage of development of human life. The initial use of diverse biophysical techniques (e.g., micropipette aspiration, atomic force and scanning ion conductance microscopy, real-time deformability cytometry) started unraveling biomechanical features of platelets that are expected to provide new explanations for physiological and pathological mechanisms. Although the impact of cytoskeletal alterations has been largely elucidated in various IPDs' pathogenesis, the understanding of their impact on biomechanical properties of platelets represents an unmet need. Regarding IPDs, improving biomechanical studies seems promising for diagnostic and prognostic implications. Potentially, these characteristics of platelets may also be used for the prediction of bleeding risk. This review addresses the current available methods for biophysical investigations of platelets and the possible implementations in the field of IPDs.

Quantifying single-platelet biomechanics: An outsider's guide to biophysical methods and recent advances.

Platelets are the key cellular components of blood primarily contributing to formation of stable hemostatic plugs at the site of vascular injury, thus preventing excessive blood loss. On the other hand, excessive platelet activation can contribute to thrombosis. Platelets respond to many stimuli that can be of biochemical, cellular, or physical origin. This drives platelet activation kinetics and plays a vital role in physiological and pathological situations. Currently used bulk assays are inadequate for comprehensive biomechanical assessment of single platelets. Individual platelets interact and respond differentially while modulating their biomechanical behavior depending on dynamic changes that occur in surrounding microenvironments. Quantitative description of such a phenomenon at single-platelet regime and up to nanometer resolution requires methodological approaches that can manipulate individual platelets at submicron scales. This review focusses on principles, specific examples, and limitations of several relevant biophysical methods applied to single-platelet analysis such as micropipette aspiration, atomic force microscopy, scanning ion conductance microscopy and traction force microscopy. Additionally, we are introducing a promising single-cell approach, real-time deformability cytometry, as an emerging biophysical method for high-throughput biomechanical characterization of single platelets. This review serves as an introductory guide for clinician scientists and beginners interested in exploring one or more of the above-mentioned biophysical methods to address outstanding questions in single-platelet biomechanics.

Firm but fair policies for staff vacations and holidays.

Paid holiday and vacations are a wonderful employee benefit for your staff. However, without some firm policies in place that anticipate and avoid problems, holidays and vacations can backfire in a medical practice and become a source of problems both for the practice and for the staff. This article provides concrete tips for reducing vacation and holiday conflicts and particularly for structuring a firm-but-fair vacation policy for new employees. It provides guidance for solo practitioners' vacation scheduling and offers a list of the most common paid holidays in professional practices today. In addition, this article offers answers to common holiday and vacation scheduling questions such as how to handle holidays that fall during a vacation and weekend holidays. Finally, this article offers specific advice for making the December holiday season a pleasant and trouble-free one for your staff.

Becoming a better delegator.

Most medical practice managers know that delegation is a useful practice management tool to streamline both personal and practice efficiency. However, delegation is often underused, misused, and misunderstood. What, precisely, should be delegated in a medical practice and to whom? What are some of the obstacles to successful delegation, and how can the astute medical practice manager identify and overcome them? Which tasks should not be delegated? Finally, why do members of the professional practice staff sometimes resist delegation? In this article, the author provides answers to these intriguing questions, as well as a useful self-quiz to rate delegation skills. In addition, the author provides strategies and sample language you can use with your staff to make your own delegated tasks and responsibilities more enthusiastically accepted.

Gathering useful opinions about the job applicant.

Getting people to talk to you about former employees' job performance can be like pulling teeth. It seems that everyone these days is afraid of saying the wrong thing and having even innocent remarks come back to haunt them. Most people say nothing. However, you can increase your chances of getting useful employment references by following a few basic guidelines. This article describes the most effective methods for conducting reference checks. It suggests who is most likely to talk with you about a former employee and how to make you approach. It provides specific questions to ask and to avoid when seeking opinions about the job applicant. This article also offers simple and easy-to-implement techniques for using reference checking to build goodwill and referrals for your practice.

Verifying a job applicant's factual credentials.

Nearly one-third of all job applicants may make unjustified claims, exaggerate, omit important information, or lie in their resumes, employment applications, or interviews. Checking credentials before you hire is the best way to protect your staff and patients and to save time, money, and effort. In this article, the author identifies the most common job application lies and pinpoints the three most common resume/application red flags. In addition, the article suggests specific information that the applicant should supply in the application to help the employer get to the truth. It offers practical advice for verifying academic credentials, employment history, and awards and honors. Finally, this article provides useful suggestions for avoiding discrimination suits in hiring practices and techniques for coping with employees who have lied to you.