Differences In Platelet Morphology Research Articles

Electron Microscope Characterization of Platelet Activation State Reveals That Wound Closure and P2Y 12 Receptors Are Major Early Determinants of Thrombus Structure in a Venous Puncture Model

Vascular damage presents in many forms and varying geometries. Nevertheless, the platelet response to endothelial damage to the blood vessel wall, be it through a prick or a full puncture wound, is thought to be staged by a qualitatively similar temporal variance in signaling agonists. For example, endothelial damage in the microvasculature is thought to be initially dominated by thrombin and later by platelet released ADP and thromboxane. The same temporal sequence in signaling has been proposed to exist in a profusely bleeding puncture wound 1. If so, platelet morphology, a gold standard of platelet activation state, could provide a strong readout of temporally distinct signaling effects. Platelet morphology has long been considered to be a reliable indicator of a strong agonist such as thrombin acting through PAR receptors that produces a rounded, pseudopod extending, degranulated, highly adhesive platelet versus weaker agonists such as ADP or thromboxane acting through P2Y 12 receptors to produce a less adhesive, somewhat rounded platelet. A testable prediction of existing hemostasis models is that temporal staging of signaling leads to temporal differences in platelet morphology within the forming/remodeling thrombus. Such hypothesized temporal differences in signaling are clinically significant as they form the basis for hypothesizing phenotypically distinct outcomes for direct acting anti-coagulants (DOACs) affecting thrombin versus anti-platelet drugs affecting P2Y 12, ADP receptors.Advances in imaging, e.g., wide area transmission electron microscopy (WA-TEM), make possible the local determination of platelet activation state with high precision 2. Taking a mouse jugular vein puncture wound model 1,2, we found that all morphologically recognized platelet activation states were present early, 1 min post puncture, with loosely bound discoid shaped platelets being the most peripherally located. For bleeding, early-stage puncture wound, these loosely adherent, low activation state platelets were located on both intravascular and extravascular thrombus aggregates. Once the puncture wound is closed, loosely adherent platelets were only found on the intravascular surfaces of the thrombus. We propose that this result is most consistent with a platelet conversion model in which new loosely adherent platelets rapidly convert to tightly packed platelets. As the thrombus remodels, 5 and 20 min post-puncture, the thrombus continued to accumulate platelets both intravascularly and extravascularly. Peripheral, discoid shaped platelets provided a source for intravascular thrombus growth. However, any subsequent extravascular thrombus growth must be due to platelet migration. Significantly, we found that cangrelor, a direct acting P2Y 12 inhibitor, stalled thrombus formation/remodeling at an early stage (Figure 1A,C,E see also ref 1,2). By WA-TEM, the accumulation of discoid-shaped, loosely adherent platelets appeared to be enhanced in a cangrelor treated 5 min thrombus (Figure 1E,F). We suggest that P2Y 12 receptors must act early in thrombus formation with the conversion of discoid to more activated platelets being most affected. In contrast, a 5-min post puncture dabigatran (DOAC) treated showed deformed architecture with inhibition of the accumulation of discoid shaped platelets/rounded loosely adherent platelets being most affected (Figure 1D,F, see also ref 2). Accumulation of degranulated platelets appeared to be lessened in both cangrelor and dabigatran treated thrombi. We propose that the simplest explanation of these results is that multiple signaling pathways act in parallel with select activation states being more dependent on one pathway than another. Clinically, our results suggest that P2Y 12 inhibitors can affect thrombus formation at early time points in addition to the late time points projected by current models.1. Tomaiuolo M., Matzko C.N., Posentud-Fuentes I., Weisel J.W., Brass L.F. & Stalker T.J. Interrelationships between structure and function during the hemostatic response to injury. Proc Natl Acad Sci USA. 116. 2243-2252 (2019).2. Rhee, Pokrovskaya I.D.,BallK., LingK., VedanapartiY., CohenJ., CruzD., ZhaoO.S., AronovaM.A., ZhangG., Kamykowski J.A., LeapmanR.D., & StorrieB. Venous puncture wound hemostasis results in a vaulted thrombus structured by locally nucleated platelet aggregates. Commun. Biol., accepted. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Quantitative Optical Diffraction Tomography Imaging of Mouse Platelets.

Platelets are specialized anucleate cells that play a major role in hemostasis following vessel injury. More recently, platelets have also been implicated in innate immunity and inflammation by directly interacting with immune cells and releasing proinflammatory signals. It is likely therefore that in certain pathologies, such as chronic parasitic infections and myeloid malignancies, platelets can act as mediators for hemostatic and proinflammatory responses. Fortunately, murine platelet function ex vivo is highly analogous to human, providing a robust model for functional comparison. However, traditional methods of studying platelet phenotype, function and activation status often rely on using large numbers of whole isolated platelet populations, which severely limits the number and type of assays that can be performed with mouse blood. Here, using cutting edge 3D quantitative phase imaging, holotomography, that uses optical diffraction tomography (ODT), we were able to identify and quantify differences in single unlabeled, live platelets with minimal experimental interference. We analyzed platelets directly isolated from whole blood of mice with either a JAK2V617F-positive myeloproliferative neoplasm (MPN) or Leishmania donovani infection. Image analysis of the platelets indicates previously uncharacterized differences in platelet morphology, including altered cell volume and sphericity, as well as changes in biophysical parameters such as refractive index (RI) and dry mass. Together, these data indicate that, by using holotomography, we were able to identify clear disparities in activation status and potential functional ability in disease states compared to control at the level of single platelets.

Ultrastructural analysis of platelets during three phases of pregnancy: A qualitative and quantitative investigation

ObjectivesIn the past, platelet morphology during normal pregnancy has not been given much attention.MethodsElectron microscopy analysis of platelets from 60 pregnant individuals (30 early pregnancy (weeks 8–14) participants and 30 late pregnancy (weeks 36–40) participants which were followed up 6–8 weeks post-partum) were compared to platelets from 30 non-pregnant individuals as well as each other to establish whether differences in platelet morphology exist during pregnancy.ResultsUltrastructural changes pertaining to the external and internal arrangements of platelets were visible. Fixated platelets showed pseudopodia formation and membrane blebbing. Increased and enlarged open canalicular system pores, pseudopodia formation, platelet spreading, and membrane blebbing were visible in vital platelets. Platelets from pregnancy were tightly packed and internal structures were different from the non-pregnant group. The internal granules showed modification in their occurrence within the cell. The α- and lysosomal granule counts were significantly increased during pregnancy while dense granule and mitochondrial numbers were significantly decreased.DiscussionThis may point to a pregnancy-specific modification. The changes in platelet ultrastructure discerned in this study attribute to the hypercoagulable state associated with pregnancy. All ultrastructural alterations associated with pregnancy persist for at least 2 months after birth.

Platelet markers correlate with glycemic indices in diabetic, but not diabetic-myelodysplastic patients with normal platelet count.

Background: Altered thrombocyte morphology and function have been reported in patients with diabetes mellitus (DM) type 2. The aim of the present study was to determine the associations between platelet morphology markers and hemoglobin A1C (HbA1c), fasting glucose (FG), hypertension and coronary heart disease (CHD) in patients with myelodysplastic syndromes (MDS) and DM, in patients with DM and in controls. Methods: This cross-sectional study included 30 cases with primary MDS with normal platelet count and non-insulin dependent diabetes, 30 non-insulin dependent diabetic patients and 30 non-diabetic, non-MDS controls matched on age and gender. Results: After adjusting for body mass index, platelet number, CHD and hypertension, HbA1c and FG were significant predictors of mean platelet volume (MPV) and platelet distribution width (PDW) in diabetic patients. There was no correlation between platelet parameters and HbA1c or FG in diabetic MDS patients. In controls, FG and hypertension predicted significant differences in platelet morphology. Platelet count correlated with platelet morphology in diabetic MDS and control groups, but not in diabetics. Conclusions: MPV and PDW are associated with glycemic indices in diabetic patients but not in diabetic MDS patients with normal platelet counts. Non-diabetic controls also exhibit FG related changes in platelet morphology. This suggests other factors inherent to bone marrow dysplasia, platelet turnover and biochemistry, or vascular environment affect platelet morphology in diabetic MDS patients even with normal platelet count. Platelet morphology in this population may be an early marker for myelodysplasia. These findings also support platelet morphology change as a marker for elevated macrovascular disease risk.

Platelet Markers Correlate with Glycemic Indices in Diabetic, but Not Diabetic-Myelodysplastic Patients with Normal Platelet Count

Background: Altered thrombocyte morphology and function have been reported in patients with diabetes mellitus (DM) type 2. The aim of the present study was to determine the associations between platelet morphology markers and hemoglobin A1C (HbA1c), fasting glucose (FG), hypertension and coronary heart disease (CHD) in patients with myelodysplastic syndromes (MDS) and DM, in patients with DM and in controls.Methods: This cross-sectional study included 30 cases with primary MDS with normal platelet count and non-insulin dependent diabetes, 30 non-insulin dependent diabetic patients and 30 non-diabetic, non-MDS controls matched on age and gender. Results: After adjusting for body mass index, platelet number, CHD and hypertension, HbA1cand FG were significant predictors of mean platelet volume (MPV) and platelet distribution width (PDW) in diabetic patients. There was no correlation between platelet parameters and HbA1cor FG in diabetic MDS patients. In controls, FG and hypertension predicted significant differences in platelet morphology. Platelet count correlated with platelet morphology in diabetic MDS and control groups, but not in diabetics.Conclusions: MPV and PDW are associated with glycemic indices in diabetic patients but not in diabetic MDS patients with normal platelet counts. Non-diabetic controls also exhibit FG related changes in platelet morphology. This suggests other factors inherent to bone marrow dysplasia, platelet turnover and biochemistry, or vascular environment affect platelet morphology in diabetic MDS patients even with normal platelet count. Platelet morphology in this population may be an early marker for myelodysplasia. These findings also support platelet morphology change as a marker for elevated macrovascular disease risk.

The role of plasma proteins and stress in the assessment of hemocompatibility.

The physiological and psychological conditions of subjects supplying blood for hemocompatibility tests significantly affect the behavior of platelets in terms of both adhesion and activation. The responses of platelets to a standard biomaterial, polyethylene (PE), were examined with blood collected from male rabbits both in basal conditions and after stress. Different media were utilized. First, platelet-rich plasma (PRP) was used to obtain a PE response to contact with platelets. Then platelets drawn from PRP were isolated and washed with Krebs-Ringer solution. One aliquot was suspended in serum (Pw-S) where fibrinogen was absent, another aliquot in Krebs-Ringer solution (Pw-KR) (in order to avoid the influence of the plasma proteins on platelets), and a third aliquot in the original plasma from which the platelets were drawn (Pw-PPP) (in order to restore the initial condition of the plasma but with washed platelets). The analysis of platelet adhesion and morphology was performed by Scanning Electron Microscopy (SEM). Differences in platelet adhesion and morphology were observed with four different media in nonstressed animals, with Pw-PPP showing a higher number and Pw-S and PW-KR lower numbers. Platelet morphology indicated low levels of activation. The platelets drawn from stressed subjects could not be counted in either PRP or PPP medium because they were fully aggregated and adhered; in contrast, in Pw-KR and Pw-S, no significant differences were found with respect to nonstressed conditions, and there was little difference in platelet morphology. All of these factors underline the role of plasma proteins, in particular fibrinogen, in the stress-induced activation of platelets.

Sex-related differences in platelet morphology in whole blood (WB) and platelet-rich plasma (PRP)

The proportion of smooth, disc-shaped platelets (D) in freshly drawn, glutaraldehyde fixed whole blood (WB) and citrated platelet-rich plasma prepared at 37°C is compared for male (N = 55) and female (N = 31) donors. Female donors have significantly more %D than male donors and the variability in %D measured on repeated occasions over a period of 3–69 months is less for female donors. WB and PRP gave similar results. The sex-related differences in %D were not related to hematocrit or to concomitant use of oral contraceptives. There were no significant sex-related differences in platelet mean volume or in platelet plasma membrane surface area as determined by the osmotic spherocyte method. However, the volume of D is smaller for male donors. It is concluded that the sex-related differences in platelet morphology do not represent intrinsic differences in platelet size or measurable total plasma membrane but represent a selective shape change activation of the larger D in the circulation of male donors. The significance of these observations for the sex-related differences in risk of cardiovascular disease and efficacy of anti-thrombotic therapy awaits appropriate prospective epidemiological studies.

Enzymatic activities, ADP-induced aggregation and survival in vivo as criteria of platelet viability during storage.

To better preserve human platelets in hyper‐citrated plasma in siliconized glassware, the authors recommend centrifugation between 7 and 10 C rather than at 4 C and the dispersion of the button of platelets either by the “propipette” method (drawing the supernatant plasma just above the deposited platelets up and down a pipette) or by a mechanical method using a Whirli mixer. With these methods of dispersion, the rise of acid nitro‐phenylphosphatase in platelets and plasma during one day of storage is less than with the use of a scraping method. The in vitro ADP‐induced aggregation is also well preserved at 4 and at 8 C. Electron microscopic studies do not reveal detectable differences in platelet morphology at these temperatures after one day of storage. The use of radiochromium for platelet tagging does not entirely support the above results but this method is probably not the best for measuring platelet viability during storage.One‐day storage at 37 C results in the non‐aggregability of platelets, conspicuous increase in platelet nitrophenylphosphatase and considerable morphological modifications of platelets, as has been observed by Firkin.7