Platelet Function In Health Research Articles

An Insight into Recent Advances on Platelet Function in Health and Disease.

Platelets play a variety of roles in vascular biology and are best recognized as primary hemostasis and thrombosis mediators. Platelets have a large number of receptors and secretory molecules that are required for platelet functionality. Upon activation, platelets release multiple substances that have the ability to influence both physiological and pathophysiological processes including inflammation, tissue regeneration and repair, cancer progression, and spreading. The involvement of platelets in the progression and seriousness of a variety of disorders other than thrombosis is still being discovered, especially in the areas of inflammation and the immunological response. This review represents an integrated summary of recent advances on the function of platelets in pathophysiology that connects hemostasis, inflammation, and immunological response in health and disease and suggests that antiplatelet treatment might be used for more than only thrombosis.

Platelets and Matrix Metalloproteinases: A Bidirectional Interaction with Multiple Pathophysiologic Implications.

Platelets contain and release several matrix metalloproteinases (MMPs), a highly conserved protein family with multiple functions in organism defense and repair. Platelet-released MMPs as well as MMPs generated by other cells within the cardiovascular system modulate platelet function in health and disease. In particular, a normal hemostatic platelet response to vessel wall injury may be transformed into pathological thrombus formation by platelet-released and/or by locally generated MMPs. However, it is becoming increasingly clear that platelets play a role not only in hemostasis but also in immune response, inflammation and allergy, atherosclerosis, and cancer development, and MMPs seem to contribute importantly to this role. A deeper understanding of these mechanisms may open the way to novel therapeutic approaches to the inhibition of their pathogenic effects and lead to significant advances in the treatment of cardiovascular, inflammatory, and neoplastic disorders.

Platelet surface marker analysis by mass cytometry

Mass cytometry is a next generation flow cytometry technology which analyzes cells one at a time (up to 1000/sec) using mass spectrometry to detect probes labeled with rare-earth metals. Rare-earth metals detected by mass spectrometry have extremely low backgrounds and can be identified with high resolution enabling the routine simultaneous detection of more than 45 probes on each cell without the need for complex compensation matrices. Here we describe a panel of 14 platelet-specific metal-conjugated antibodies (targeting cluster of differentiation [CD] 9, CD29, CD31, CD36, CD41, CD42a, CD42b, CD61, CD62P, CD63, CD107a, CD154, glycoprotein [GP] VI and activated integrin αIIbβ3) and methods for staining and analysis of platelets by mass cytometry. High dimensional clustering algorithms, which take into account the levels of all 14 markers detected by mass cytometry on each cell, allow identification of platelet subpopulations not previously appreciated. We previously reported that platelet heterogeneity identified by mass cytometry appears similar across healthy donors and consistent over time. High dimensional analysis revealed the presence of a platelet subpopulation with significantly higher levels of surface expression of activated GPIIb-IIIa and P-selectin suggesting this subpopulation may play a greater role in thrombus formation than other platelet subpopulations. Thus, analysis by mass cytometry of platelet heterogeneity and subpopulations may suggest distinct biological roles for different platelet subpopulations and may be useful in evaluating inherited or acquired platelet disorders and platelet function in health and disease.

Platelet Contraction Force As a Biophysical Biomarker for Bleeding Risk in Patients with Immune Thrombocytopenia

Background: Immune Thrombocytopenia Purpura (ITP) is defined by a low platelet count in the absence of any known causes and affects over 4,000 US children and 8,000 adults each year (Terrell et al, 2010). Deciding when and how to treat these patients remains difficult as there is no biomarker or diagnostic test that identifies which patients will self-resolve and which are at risk for major bleeding and life-threatening intracranial hemorrhage (~10%). In addition, the medications used to treat ITP all have significant side effects, forcing clinicians to balance risks associated with bleeding and medication. As such, an ongoing debate in the field of clinical hematology centers around which patients require therapy (Flores & Buchanan, 2013)(Cooper 2017)(Gralnek 2008). Here, we show that a new quantitative measurement of platelet function, which is independent of known platelet biomarkers (Myers et al, 2017), has diagnostic potential in identifying bleeding risk in ITP patients. Specifically, using high-throughput platelet contraction cytometry (PCC) measurements of individual platelet forces (Myers et al, 2017), we found that low platelet forces strongly correlate with bleeding symptoms. Unlike existing tests of platelet function that use bulk measurements, our test operates at the single cell level and therefore does not depend on the platelet count, enabling a direct comparison of individual platelet function in health versus disease states. In the broad context of overall function, our findings of impaired force generation agree with previous research demonstrating that impaired platelet function, and not low platelet count, correlates with bleeding in ITP patients (Frelinger et al, 2015). In addition, as the first demonstration of a correlation between single cell force and disease, our novel approach may represent an entirely new class of “physics-based” diagnostics.Platelet Contraction Cytometry (PCC): Within the PCC, a single platelet attaches, spreads, and applies contractile force to a pair of fibrinogen microdots that are attached to a moveable, spring-like, surface. Since the applied platelet contractile force is directly proportional to the microdot displacement, the force is calculated from a single fluorescence image of the platelet. Using microfabrication technology, thousands of microdots are created on a single device to enable high-throughput measurements in tightly controlled mechanical, biochemical, and shear microenvironments (Fig 1). Here, gel-filtered platelets from patients are activated (1U/mL thrombin), plated on the device with a moderately stiff surface (75 kPa), and measured as described previously (Myers et al, 2017).Results: In a cohort of ITP patients (n = 27), we observed that patients with bleeding and/or bruising symptoms exhibited significantly lower average platelet contraction forces than asymptomatic patients regardless of platelet count. Using an average force cutoff value of 26nN, we found that low forces identified bleeding in ITP with 100% sensitivity and 89.4% specificity. From a mechanistic perspective, our preliminary data also suggests that mean platelet volume does not correlate with platelet force or function, although further studies on a single platelet level are needed to confirm this result. While this data indicates that patients with bleeding symptoms have platelets with low forces, it is unclear whether low forces correlate with bleeding or whether the patient always has had low platelet forces that therefore render them susceptible to bleeding. Our preliminary prospective data suggests that low platelet forces correlate with the bleeding symptoms themselves and return to higher values when bleeding symptoms cease.Importantly, as our approach enables “single-cell” examinations of platelet forces, we identified different platelet subpopulations unique to ITP. Healthy individuals have a distribution of contractile forces with a single peak, while ITP patients tend to have two prominent peaks, one with a lower contractile force and one with a higher one. Surprisingly, patients with bleeding symptoms only have a low force peak, suggesting that the lack of highly contractile platelets may be a biophysical biomarker for bleeding (Fig 2). Ongoing studies are focused on using this finding to further improve specificity as well as elucidating the mechanistic underpinnings of low platelet contraction in ITP. DisclosuresNo relevant conflicts of interest to declare.

Matrix Metalloproteinases and Platelet Function.

Platelets contain and release several matrix metalloproteinases (MMPs) and their tissue inhibitors of matrix metalloproteinases (TIMPs), including MMP-1, -2, -3, -9, and -14 and TIMP-1, -2, and -4. Although devoid of a nucleus, platelets also synthesize TIMP-2 upon activation. Platelet-released MMPs/TIMPs, as well as MMPs generated by other cells within the cardiovascular system, modulate platelet function in health and disease. In particular, a normal hemostatic platelet response to vessel wall injury may be transformed into pathologic thrombus formation by the release from platelets and/or by the local generation of some MMPs. Moreover, platelets may localize the production of leukocyte-derived MMPs to sites of vascular damage, contributing to atherosclerosis development and complications and to arterial aneurysm formation. Finally, the interaction between platelets and tumor cells is strongly influenced by MMPs/TIMPs. All these mechanisms are emerging as important in atherothrombosis, inflammatory disease, and cancer growth and dissemination. Increasing knowledge of these mechanisms may open the way to novel therapeutic approaches.

Characterization of the aggregation responses of camel platelets

Despite evidence of active hemostasis, camel platelets barely respond to common aggregating agents at standard doses used for human platelet aggregation. The purpose of the study was to find out whether camel platelets can be activated by high doses or combinations of aggregation agonists, and to characterize the receptor that mediates the aggregation response to adenosine diphosphate (ADP), the most potent agonist for camel platelets known so far. Aggregation studies were performed with platelet-rich plasma (PRP) in response to multiple doses or combinations of ADP, epinephrine (EPN), collagen, and arachidonic acid (AA). Aggregation responses to ADP were performed before and after the addition of the ADP receptor (P2Y12) antagonist Clopidogrel. Camel platelets responded to ADP at doses higher than the standard dose for human platelets, and to combinations of EPN and other agonists, while no aggregation was elicited with EPN or AA alone. Clopidogrel blocked the ADP-induced aggregation responses in a dose-dependent fashion in vitro. Camel platelet aggregation can be activated by increasing the dose of some agonists such as ADP, but not AA or EPN. Irreversible aggregation of camel platelets could also be triggered by a combination of EPN and ADP, and collagen and AA. Inhibition with clopidogrel suggests that camel platelets express the ADP receptor, P2Y12. Understanding platelet function in camels will add to the understanding of platelet function in health and disease.

Platelet Function in Health and Disease: from Molecular Mechanisms, Redox Considerations to Novel Therapeutic Opportunities

Increased oxidative stress appears to be of fundamental importance in the pathogenesis and development of several disease processes. Indeed, it is well known that reactive oxygen species (ROS) exert critical regulatory functions within the vascular wall, and it is, therefore, plausible that platelets represent a relevant target for their action. Platelet activation cascade (including receptor-mediated tethering to the endothelium, rolling, firm adhesion, aggregation, and thrombus formation) is tightly regulated. In addition to already well-defined platelet regulatory factors, ROS may participate in the regulation of platelet activation. It is already established that enhanced ROS release from the vascular wall can indirectly affect platelet activity by scavenging nitric oxide (NO), thereby decreasing the antiplatelet properties of endothelium. On the other hand, recent data suggest that platelets themselves generate ROS, which may evoke pro-thrombotic responses, triggering many biological processes participating in atherosclerosis initiation, progression, and complication. That oxidative stress may alter platelet function is conceivable when considering that antioxidants play a role in the prevention of cardiovascular disease, although the precise mechanism accounting for changes attributable to antioxidants in atherosclerosis remains unknown. It is possible that the effects of antioxidants may be a consequence of their enhancing or promoting the antiplatelet effects of NO derived from both endothelial cells and platelets. This review focuses on current knowledge regarding ROS-dependent regulation of platelet function in health and disease, and summarizes in vitro and in vivo evidence for their physiological and potential therapeutic relevance.

Possible Role for Platelet Insulin Receptors in Modulating Platelet Function in Health and Diabetes Mellitus

Binding studies have shown that human platelets contain binding sites for insulin with a surface density similar to that described for other cells.(1) Evidence for a reduced number and affinity of human platelet membrane insulin binding sites in non-insulin dependent diabetes mellitus (NIDDM) has been provided.(2) However, the influence of insulin and insulin receptors on platelet function has not been completely investigated and clarified. Falcon et al(3) reported phosphorylation of a subunit of the insulin receptor on platelets in response to insulin, but no alterations were detected in cAMP formation or degradation, inositol phosphate formation or phosphorylation of proteins other than the receptor itself. On the other hand Trovati et al(4) found reduced platelet aggregation responses to ADP, PAF, epinephrine, collagen and arachidonate in the presence of 40 µU/ml insulin. Their experience with an euglycemic-hyperinsulinemic clamp provided some further in vivo evidence to support the above mentioned findings. Platelet thromboxane A(2) formation was not altered in the presence of the hormone.

Sialic Acid: Its Importance to Platelet Function in Health and Disease

Platelets are known to contain sialic acid, most of which resides on glycoprotein carbohydrate side-chains. A large proportion of the platelet sialic acid is neuraminidase-labile. The sialic acid of human platelets is of the N-acetyl neuramininic acid type and is implicated in platelet functional events, such as aggregation and adhesion as well as contributing to the platelet's electrophoretic mobility. In this respect the platelet has an overall electronegative charge which is predominately the result of the carboxyl group of the sialic acid moiety. Platelet subpopulations have been isolated with differing amounts of sialic acid and cleavage of platelet sialic acid by neuraminidase is associated with loss of platelets from the circulation. This has relevance to platelet aging in vivo where removal of platelet sialic acid may reveal 'senescence antigens' which could facilitate platelet 'uptake' by the reticuloendothelial system. The importance of platelet sialic acid status has been described in certain platelet disorders such as some thrombocytopenias as well as particular disease states including malignancies and atherosclerosis.

Blood Platelet Function and Medicinal Chemistry

One hundred years after the discovery of platelets in blood by Giulio Bizzozero ( Arch Pathol Anat 1882;90: 261), these extraordinarily explosive and versatile cells are now recognized not only as integral components of hemostasis but also as participants in the pathogenesis of atherosclerosis, vascular disease, and perhaps even malignant transformation and proliferation. In this volume, Lasslo has brought together current knowledge of some of the most exciting areas of platelet physiology, biochemistry, and pharmacology. The chapters contain discussions of the role of platelets in cardiovascular disease, platelet ultrastructure, normal and abnormal platelet function, endothelial physiology, and interactions between platelets and artificial surfaces and between platelets and medicinal and chemical agents. The title of the book is somewhat misleading, because the scope of the work is much broader than implied by its focus on medicinal chemistry; it is, in fact, a general review of platelet function in health and disease. The

Progress in Hematology, vol 7.

As in previous volumes of the Progress in Hematology series, Brown and Moore have edited a collection of review articles from expert contributors discussing current developments in hematology. The subject material is extensive in scope, and there is a morsel to please every palate. For those oriented to physiology, there are comprehensive chapters dealing with the contribution of red blood cells (RBC) to blood rheology, the interrelationship between RBC metabolites, hemoglobin, and the oxygen-equilibrium curves, a thorough discussion of the unstable hemoglobins, and an analysis of structure and function of cell surface receptors. Timely topics include food iron absorption and its relation to iron supplementation of food, the hematopoietic action of androstanes, and the current understanding of the kallikrein-kinin system of human plasma. The clinically oriented will find pleasure in the chapters dealing with platelet function in health and disease, disorders of phagocytic cell function, and with the splendidly