Changes of platelet morphology, ultrastructure and function in patients with acute ischemic stroke based on super- resolution microscopy

doi:10.21203/rs.3.rs-2344065/v1

There still require more detail information of platelet ultrastructure and antiplatelet drug choices in acute ischemic stroke (AIS) patients. These are restricted by microscopic limitations and sensitivity of platelet. The new super-resolution microscope (SIM) can accurately, quickly analyze platelet. We applied SIM to observe the morphology and ultrastructure of platelets with AIS patients in four different states. Analyzing SIM images to quantification the change of dense granules and α granules in platelets. Testing platelet factor 4 (PF4) to reflect platelet granule function. We speculated that the diameter of platelets, average size of granules, area% of granules per field and mean area of granules per platelet in AIS patient were lower than healthy people in rest stage. Under activated state, α granule of AIS patients showed a pattern of parenchymatousfluorescent masses. Platelet granule function was suppressed at rest stage and showed hyper-responsiveness after stimulation in AIS patients. 2MeSamp has a stronger inhibition effect to α granules of platelets than aspirin. According to the results, there are heterogeneity in the structure and function of platelets in AIS patients. Additionally, analyzing platelets based on SIM could be used as a new method to indicates the onset of AIS and assess antiplatelet drugs.

Biological sciences/Cell biology

Health sciences/Diseases

Health sciences/Neurology

Acute ischemic stroke (AIS) is the second leading cause of death worldwide, the most fatal sudden disease after heart disease, and the leading cause of disability [1, 2]. By 2030, stroke-related disability is projected to be the fourth most important cause of disability-adjusted life years in developed countries [3]. AIS refers to the sudden termination of blood supply to the brain tissue when the cerebral blood flow is below the ischemic threshold (10–25% of baseline)[4], causing irreversible nerve cell damage in the corresponding ischemic area and eventually lead to nerve function impairment. Intracranial atherosclerosis is the most common cause of AIS, and platelets play an inevitable role in subsequent vascular blockage[5].

Under physiological conditions, platelets play the role of homeostasis, inflammation and immune response. On the contrary, it is over activated to form thrombosis in some pathological situations such as ischemic stroke or acute myocardial infarction[6, 7]. In AIS patients, platelet surface is exposed to circulating thrombin and von Willebrand factor (vwf) [8]. During platelet activation, platelets ultrastructure named dense granules and alpha granules can release adenosine diphosphate (ADP), serotonin (5-HT) and β-thromboglobulin (β-TG), platelet factor 4 (PF4) respectively [9]. With the participation of these granules, platelet throughout adhesion as well as aggregation, and eventually forms thrombosis to block the cerebral blood vessel, leading to the occurrence of AIS [2]. Some studies have pointed out that platelets are more likely to be activated with the increase of age, which may contribute to the higher incidence of AIS in elder people [10]. As highly reactive blood cells, platelets have undergone significant changes in their ultrastructure in some diseases, such as changes in platelet microtubules and mitochondria in patients with ovarian cancer [11]. Another study noted a significant reduction in the dense granules of platelets in patients with Hermansky-Pudlak syndrome [12]. In view of the tightly connect between AIS and platelets, more details of the changes of platelet morphology, ultrastructure and granule function in AIS patients will be vital for the diagnosis and treatment of this disease.

Antiplatelet drugs are widely used for secondary prevention of AIS. According to the 2018 American Heart Association/American Stroke Association and 2019 BMJ Clinical Practice guidelines, early combined aspirin and clopidogrel should be used in patients with mild stroke and transient ischemic attacks (TIA), but long-term use is not recommended[13, 14]. As a thromboxane A2 (TXA2) inhibitor, aspirin irreversibly inhibiting platelet cyclooxygenase-1 (COX-1) to prevent the formation of TXA2[8]. Clopidogrel acts as an inhibitor of ADP receptor which activates platelets by binding to two protein receptors on platelets (P2Y1 and P2Y12) [15]. However, the majority (82 to 87%) of patients taking aspirin alone are not protected from further vascular events [16]. It also has been suggested that clopidogrel may have a benefit over aspirin in patients with ischemic cerebrovascular disease (CVD) [17]. Which drug is better for AIS patients is still controversial and it may need more detail study of platelets to support. Therefore, we compared the different effects on platelet subcellular structure as well as release function between the two drugs.

In most studies, platelet function was measured by light transmission aggregation assay (LTA), flow cytometry, thrombus elastography and other methods[18]. SIM as a new kind of optical imaging technology, with high resolution, rapidity and unbiased data, can provide convenient and reliable analysis of platelet ultrastructure. Existing research have applied SIM to investigate platelet ultrastructure in illness state, and provided new ideas for disease diagnosis[11, 12, 19]. Since the dense granules and α granules of platelets are critical in platelet activation of AIS patients, the two granules with four different states of platelets in AIS patients were observed and analyzed by SIM, including (1) rest; (2) activate; (3) activation after 2MeSAMP treatment; (4) activation after Aspirin treatment. 2MESAMP and Aspirin are two in vitro antiplatelet drugs that inhibit cyclooxygenase-1 (COX-1) and P2Y12 receptor, respectively. Ultrastructure observation and analysis of platelets by SIM could offer more details of platelets in AIS patients. Through the changes of platelet ultrastructure in the elderly might monitor the occurrence of this disease. Meanwhile, the reactivity of platelets to different antiplatelet drugs could be evaluated to supply reference for clinical individualized medication.

No significant changes of rest platelet morphology were observed in AIS patients by SIM

The CD63 and vwf structures were marked by immunofluorescence staining in dense granules and α granules of platelets, respectively. We observed morphology and ultrastructure of platelets in AIS patients and healthy control group with the help of SIM (Fig. 1). Under SIM observation, the rest platelets of AIS patients were disc-shaped, with smooth and neat edges, and there were no obvious morphological changes with healthy control (Fig. 1). Dense granules and α granules with important release function are organelle structures in platelets. It can be clearly seen that the two kinds of granules showed punctuate distribution scattered in the platelet, with no aggregation between the granules by SIM (Fig. 1). Subsequently, we observed the two granules of platelets in AIS patients, and there was no obvious heterogeneity in morphology between AIS patients and healthy controls (Fig. 1).

Quantitative analysis of SIM images showed the diameter and average size of CD63 and vwf of single platelet reduced in AIS patients

Although no morphological differences were observed, platelet diameter was calculated from totally 1900 platelets in AIS patients and healthy subjects for comparison by SIM images (Fig. 2a-b). In order to avoid the bias caused by the influence of age on platelet, we selected the elderly healthy group (≥ 60 years old) and the young healthy group (18 ~ 35 years old) as the healthy control group. Information on age, sex, National Institute of Health stroke scale (NHISS) score, MRI results, past history, medication history, platelet count, mean platelet distribution width, mean platelet volume, and ratio of large platelets between patients and controls are shown in Table 1 (Table 1). A total of 5372 platelets from 7 patients and 12

Table 1

**Characteristics of AIS patients and healthy control.** Patient(P), Elder health (EH), Young health (YH), National Institute of Health stroke scale(NIHSS), National Institute of Health stroke scale (MRI), Platelet distribution width(PDW), mean platelet volume(MPV), platelet large cell ratio (P-LCR).
Number	Sex	Age	NIHSS score	MRI	Complication	Drug administration	Platelet number (125 ~ 350*10⁹)	PDW (15.5 ~ 18.1fL)	MPV(9.4 ~ 12.5fL)	P-LCR (17.5 ~ 24.3%)
P1	M	58	0	Lacunar infarction	hypertension、PU	/	243	16.7	12.3	10.4
P2	F	71	4	Right basal ganglia infarction	hypertension	/	234	14.1	11.2	35
P3	M	59	3	Coronal region infarction	hypertension、CKD	/	232	12.9	11.1	33.8
P4	M	54	6	Right cerebellar infarction	hypertension、CKD	nifedipine	158	16.1	12.2	43.4
P5	F	85	3	Left basal ganglia infarction	hypertension	/	217	16.5	9.4	21.9
P6	M	62	2	Right parietal lobe infarction	/	/	290	10.5	9.9	23.9
P7	M	65	4	Left parietal lobe infarction	/	/	148	15.6	11.6	38.3
EH1	M	74	/	/	/	/	223	12.4	10.5	29.3
EH2	F	62	/	/	PD	madopar	203	13.2	11.7	37.8
EH3	M	70	/	/	APB	metoprolol	288	10.9	9.9	24
EH4	M	67	/	/	/	/	206	13	10.7	30
EH5	F	65	/	/	/	/	163	17	13	48.3
EH6	M	73	/	/	/	/	201	11	9.9	23.8
YH1	F	24	/	/	/	/	253	11.1	8.9	18.5
YH2	M	25	/	/	/	/	160	10	8.9	17.8
YH3	M	22	/	/	/	/	224	10.7	9	18.9
YH4	M	26	/	/	/	/	240	10.6	9.1	18.7
YH5	F	21	/	/	/	/	209	8.5	7.9	19
YH6	M	29	/	/	/	/	293	10.6	9.1	18.7

healthy controls (EH and YH) were selected. Finally, the average values of all samples were calculated. The mean platelet diameter of the patients was shorter than that of the elderly healthy group and the young healthy group (3.30 ± 0.40 µm vs 3.60 ± 0.29 µm, P = 0.151;3.30 ± 0.40 µm vs 4.03 ± 0.32 µm, P = 0.004) (Fig. 2b), and the results were significantly different. Otherwise, there were differences between elderly healthy group and the young healthy group (3.60 ± 0.29 µm vs 4.03 ± 0.32 µm, P = 0.0356).

Similarly, we quantitatively analyzed the image information of platelet dense granules and α granules obtained by SIM. Due to the heterogeneity of platelets, a total of 8171 platelets were selected and 8 parameters were measured: The number of CD63 and vwf, the average size of CD63 and vwf, the mean area of CD63 and vwf per platelet, as well as the CD63 and vwf area% in each field. In addition to the number of granules, the average size of platelet dense granules and α granules, the mean area of platelet within each platelet, and the area% in each field were significantly reduced in AIS patients (Fig. 2). The CD63 average size of AIS patient was significantly lower than healthy control including EH and YH (0.69 ± 0.32 µm² vs 1.45 ± 0.79 µm², P = 0.038; 0.69 ± 0.32 µm² vs 1.48 ± 0.60 µm², P = 0.0111; Fig. 2e). Meanwhile, there were no difference between EH and YH (1.45 ± 0.79 µm² vs 1.48 ± 0.60 µm², P = 0.942, Fig. 2e). The mean area of CD63 per platelet in AIS patients was significantly lower than that in EH group, but there was no difference between patient group and YH group (24.88 ± 10.88 µm² vs 96.20 ± 49.16 µm², P = 0.0032; 24.88 ± 10.88 µm² vs 38.89 ± 37.61 µm², P = 0.364, Fig. 2f). In the meantime, this parameters in EH group was higher than YH group (96.20 ± 49.16 µm² vs 38.89 ± 37.61 µm², P = 0.0467, Fig. 2f). The CD63 area% in each field in AIS patient was smaller than healthy control including EH and YH (0.3 ± 0.2 vs 1.0 ± 0.6, P = 0.0159; 0.3 ± 0.2 vs 0.9 ± 0.7, P = 0.0541; Fig. 2g). When assessing vwf, the other ultrastructure of platelets, there were similar results like CD63. Firstly, the average size of vwf in AIS patient was also lower than healthy control including EH and YH (1.54 ± 0.44 µm² vs 2.87 ± 1.75 µm², P = 0.035; 1.54 ± 0.44 µm² vs 1.70 ± 0.64 µm², P = 0.6084; Fig. 2j). Meanwhile, there were no difference between EH and YH (2.87 ± 1.75 µm² vs 1.48 ± 1.70 ± 0.64 µm², P = 0.0649, Fig. 2j). Then, the mean area of vwf per platelet in AIS patients was lower than that in EH group and YH group, and there was significantly difference between patient group and EH group (765.03 ± 488.57 µm² vs 1670.91 ± 439.53 µm², P = 0.0051; 765.03 ± 488.57 µm² vs 1004.78 ± 902.08 µm², P = 0.4452, Fig. 2k). In the meantime, this parameters in EH group was higher than YH group but with no difference (1670.91 ± 439.53 µm² vs 1004.78 ± 902.08 µm², P = 0.0649, Fig. 2k). Finally, the vwf area% in each field in AIS patient was smaller than healthy control including EH and YH (1.29 ± 0.44 vs 4.26 ± 2.86, P = 0.0195; 1.29 ± 0.44 vs 4.51 ± 1.78, P = 0.0007; Fig. 2l). The other two parameters of CD63 number and vwf number had no statistical difference (Fig. 2c, i).

α granule in platelet of AIS patients changed to a pattern of parenchymatous fluorescent masses upon stimulation of TH observed by SIM

Platelets were stimulated with 0.5U thrombin (TH) in healthy subjects and AIS patients, and the morphological changes of platelets, platelet dense granules and α granules were observed by SIM at different time periods of 0min, 5min and 1h (Fig. 3a). 5 minutes after activation, platelets gradually spread out from the disk and extending pseudopodia. α granules began to aggregate and adhere to each other in both healthy people and AIS patients (Fig. 3a, S1). Multiple platelets gathered in the same area to form blood clots, and the shape of a single platelet could not be recognized after 1h activation (Fig. 3a). At this time point, α granules showed morphological differences between AIS patients and healthy people. In AIS patients, α-granules accumulate together to form large fluorophores with few diffuse particles around them (Fig. 3c). The formation of α granules with less gap was closer to the structure of a pattern of parenchymatous fluorescent masses. However, in healthy people, no matter EH or YH, there are more gaps between α granules to form honeycomb texture structure like masses, and more granular parts are scattered around the masses (Fig. 3a, c). There was no significant difference in the morphology of dense granules at 0min, 5min and 1h after thrombin activation (Fig. 3b).

2MeSamp inhibited the formation of parenchymatous fluorescent masses morphology of platelet α granules in AIS patients

2MeSAMP and Aspirin are two kinds of antiplatelet drugs that inhibit ADP receptor and COX-1 receptor, respectively. Platelets of AIS patients were treated with 2MeSAMP and Aspirin at concentrations of 10µM and 53µM in vitro, respectively, and then activated with 0.5U TH for 1h. More single platelets could be observed after treated by 2MeSAMP which belongs to ADP antagonist in the wide field, compared with direct or aspirin treatment (S2). In the meantime, the α granules of platelets in AIS patients changed from parenchymatous fluorescent masses morphology to honeycomb texture structure like masses after treatment with 2MeSAMP (Fig. 4a). On the other hand, this alteration hasn’t been observed after aspirin treated (Fig. 4a). This phenomenon showed that 2MeSAMP inhibit the formation of parenchymatous fluorescent masses morphology of platelet α granules in AIS patients. And it indicated that a pattern of parenchymatous fluorescent masses morphology of α granules in platelets may represent a higher level of activation. No morphological changes were observed in dense granules (Fi 4b, S3).

Release level of PF4 was higher in AIS patient upon TH stimulation and treatment with 2MeSamp inhibited PF4 releasing

PF4 is a protein released by α granules. We measured the release of PF4 to evaluate the function of α granules between AIS patients and healthy controls. The levels of PF4 in EH were 233.95 ± 4.57ng/ml, 493.77 ± 16.27ng/ml and 1992.25 ± 57.77ng/ml at rest, 5min after activation and 1h after activation, respectively (Fig. 5a). The levels of PF4 in AIS patients were 147.15 ± 2.51ng/ml, 468.82 ± 27.02g/ml and 1272.48 ± 80.91ng/ml at rest, 5min after activation and 1h after activation, respectively (Fig. 5b). The PF4 releasing of AIS patient was significantly lower than healthy group in rest stage (147.15 ± 2.51ng/ml vs 233.95 ± 4.57ng/ml, P < 0.0001; Fig. 5c). Due to the heterogeneity of platelets, it is not reasonable to directly compare the release of PF4 from α granules in platelets with AIS patients and healthy controls. Therefore, the platelet release from AIS patients and healthy controls were normalized and then compared. The fold change of PF4 release after 5min and 1h activation in patients and healthy subjects were (3.19 ± 0.18 vs 2.11 ± 0.07, P = 0.0007; Fig. 5d) and (8.65 ± 0.55 vs 8.52 ± 0.25, P = 0.724; Fig. 5d). The release of PF4 from platelets in AIS patients after treatment with 2MeSamp and Aspirin in vitro and the ratio of PF4 release with direct activation can reflect the effect of the inhibitor. Regardless of 5min or 1h activation, 2MeSamp had a greater inhibitory effect on PF4 release than Aspirin (0.67 ± 0.09 vs 1.67 ± 0.19, P = 0.0012; 0.57 ± 0.08 vs 0.02 ± 0.87, P = 0.003; Fig. 5f).

Electron microscopy can see cells at the single-nanometer level, but sample preparation is time-consuming and requires experienced researchers to identify specific organelles. SIM, a new imaging technique, employs immunofluorescence to label the subcellular structure of cells, which has the advantages of high resolution, precise observation, short time consuming, less damage to samples and easier imaging. The advantages of SIM enable us to better examine the morphology and ultrastructure of platelets in AIS patients. It is easier to obtain more accurate picture information, including platelet morphology, the structure of platelet dense granules and α-granules. At the same time, we obtain a large number of picture information to process and conduct parameter analysis. Through the parameter analysis of the image obtained by SIM, we primly found that the mean platelet diameter of AIS patients was smaller than that of healthy group. Subsequently we analyzed the number, size and area of the α granules and dense granules in platelets. In addition to the number, the average size, area in single platelets and the area% of the two granules in AIS patients’ platelets were lower than those in healthy controls. Previous imaging methods can only count the number of granules for which they were relatively imprecise, lacking the evaluation of the size and area of them. The high resolution of SIM makes it possible to process the image information to obtain the corresponding parameters of the size and area of platelet dense granules and α granules. Therefore, with the application of SIM, we can objectively analyze the structural changes of the two kinds of granules in AIS, especially when the granule number have no difference. Therefore, the analysis of platelet ultrastructure based on SIM can be considered as a new method to detect AIS in the future. By recognizing people with abnormal platelets early, we could reduce the risk of AIS in these populations through early and appropriate intervention.

In AIS patients, the size and area of platelet dense granules and α granules were decreased compared with healthy controls based on analyzing of SIM images (Fig. 2). These alterations indicated that ultrastructure in platelet has changed during AIS processing. The mechanism of this change may be due to the highly sensitive state of circulating platelets in AIS patients, and the body initiate negative feedback regulation to reduce the abnormal activation of platelets. Although the number of dense granules and α granules in AIS patients has not changed significantly, but the structure has changed in response to the hypersensitive state of platelets. The morphological differences may affect the granules release function to some extent. This can be proofed by lower PF4 release in AIS patients which indicated that the function of platelets were inhibited in rest state (Fig. 5d).

However, the platelets of AIS patients present a hyperresponsiveness after stimulating. This is not only reflected in ultrastructure alteration based on SIM, but also embodied in greater release function tested by ELISA. When platelets were activated, it was observed that platelet α granules in AIS patients changed from dispersed round granules to a pattern of aggregated parenchymatous masses while healthy control group showed honeycomb texture structure like masses (Fig. 3a, c). Meanwhile, the fold change of PF4 release were significantly higher in AIS patients than in healthy controls at 5min of activation (Fig. 5d). After treatment with the antiplatelet drug 2MeSAMP, α granules of activated platelets in AIS patients changed from a pattern of parenchymatous fluorescent masses to honeycomb texture structure like masses following decreased PF4 releasing (Fig. 4a, 5b). Therefore, we believe that the parenchymatous fluorescent masses of α granules is a manifestation of greater activation compared with the honeycomb texture structure like masses. The different morphology of α granule might correlation with the hyperresponsiveness of platelets. Consequently, we considered that platelets in AIS patients were hyperactivity but suppressed in rest stage and alter to hyperresponsiveness after being stimulated. This means when subjected to the same degree of stimulation, the platelets of AIS patients are more easily to be activated and α granules are more closely bound than healthy people. The conclusion is also consistent with previous studies [20, 21]. α granules contain hundreds of proteins and numerous coagulation factors which play an important role in platelet aggregation and adhesion to the damaged site [2]. Therefore, we speculate that the more tightly clumped α granules in AIS patients may indicate the platelets play a more powerful role in aggregation and adhesion after stimulation than healthy people.

The hypersensitive and hyperresponsiveness of platelets in AIS patients could be inhibited by antiplatelet drug 2MeSAMP. When treated with 2MeSAMP, an ADP receptor antagonist with in vitro activity, α granules of activated platelets in AIS patients could be depolymerized from a pattern of parenchymatous fluorescent masses mass to honeycomb texture structure like masses (Fig. 4a). In contrast, treatment with aspirin that is a COX-1 inhibitor showed no significant change in platelet α granules. We considered that this may represent a stronger inhibitory effect of 2MeSAMP on platelets, in the absence of metabolic effects in vivo. Besides, the fact that the release of PF4 from α granule was lower after treatment with 2MeSAMP than aspirin treatment confirmed the consideration (Fig. 5f). We can conclude that 2MeSAMP not only suppresses the combination of P2Y12 receptor, but also change the ultrastructure and function of α granule in activated platelets. Several studies have demonstrated that P2Y12 receptor has an influence on granule release, fibrinogen receptor activation and TXA2 production besides function on ADP receptor[22, 23]. As a consequence, antagonizing P2Y12 receptor means exerting antiplatelet effects in a variety of ways which may show a better efficacy of antiplatelet. In view of 2MeSAMP belongs to P2Y12 receptor antagonist, this may indicate that P2Y12 receptor antagonist is more effective than COX-1 inhibitor in vitro combining our results. A number of clinical studies have given opinion that P2Y12 receptor antagonist was more effective in protecting patients with AIS or coronary heart disease than COX-1 inhibitor[24, 25]. However, there are some studies have a contrary opinion that clopidogrel showed a higher risk of recurrent stroke in comparison with aspirin[26]. This variant may result from antiplatelet drug resistance, racial difference and individual differences of patients. In virtue of SIM, we observed more detail ultrastructure change in platelets after treatment of 2MeSAMP and aspirin. The results indicate antagonize P2Y12 receptor may have a greater influence in inhibiting platelets activation compared to blocking COX-1 and supply a new perspective in assessing the effectiveness of antiplatelet drug the same time.

Although platelets play an important role in AIS disease, there is still thirsty of knowledge on detail information of platelet ultrastructure and its change in AIS patients. Meanwhile, whether choice P2Y12 receptor antagonist or COX-1 inhibitor in the later stages of AIS is still controversial. On the strengthen of SIM, we found the ultrastructure of platelets in AIS patients is heterogeneous either in a resting state or an activated state. Otherwise, 2MeSAMP has a stronger inhibitory effect to α granule than aspirin in vitro. In the future, we may use SIM to detect the platelets in high-risk population of AIS. And P2Y12 receptor antagonist could be considered as a more effective antiplatelet drug for long-term monotherapy.

Materials and reagents

Thrombin, Aspirin and 2-methyl-thioadenosine-monophosphate (2MeSAMP) were purchased from Sigma (America). We made a purchase of 8%Paraformaldehyde Aqueous Solutuon (PFA) from Electron Microscopy Sciences (America). We bought Anti-CD63 (ab8219), Alexa Fluor dyes 488 (ab150113) and Alexa Fluor dyes 647 (ab150079) from abcam (England). Anti-human Von Willebrand Factor (vwf) was purchased from Dako(Denmark). Human platelet factor 4(PF4) ELISA Kit were purchased from Cusabio (China).

Patients’ selection

Patients with a diagnosis of AIS according to Chinese guidelines for diagnosis and treatment of acute ischemic stroke 2018 were recruited from Wuhan Puai Hospital. Other inclusion criteria were: (1) over 18 years old without pregnancy or suckling; (2) the incidence time of ischemic stroke was 24 hours at the most; (3) all patients received computed tomography (CT)or magnetic resonance imaging (MRI) of brain and responsible focus have been observed; (4) focal neurological deficits as well as global neurological deficits attributed to cerebral ischemia. Exclusion criteria was made of : (1) cerebral hemorrhage or other pathology situations including cerebral vascular malformation, brain tumor and encephalopyosis were found in brain CT or MRI; (2) the patients who have taken any type of antiplatelet drugs or medicines influence the function of platelets in 14 days; (3) history of atrial fibrillation, severe hepatic insufficiency, end-stage renal disease, autoimmune diseases, malignant tumor, blood system disease; (4) gastrointestinal bleeding or major surgery during 3 months; (5) administration of intravenous anticoagulants or thrombolytics within 1 week; (6) having a fever or infectious disease in 2 weeks.

Control group choosing

Age may influence the function and structure of platelets[27], consequently we choose control groups divided into young healthy (YH,18 ~ 35 years old) and elderly healthy (EH ,over 60 years old) the same time in order to avoid age confounding factor. Control group came from neurology ward of Wuhan Puai Hospital from December 17, 2021 to June 21, 2022. Both of the controls need to meet these requirements that mentioned above except suffering an acute ischemic stroke.

Platelet preparation

Samples were collected from December 17, 2021 to June 21, 2022. Human whole blood was collected into a 1:9 solution of sodium citrate from neurology ward of Wuhan Puai Hospital. The collected blood was centrifuged at 200×g for 12 min in 6 hours to obtain platelet-rich plasma (PRP) which would be treated with either 10uM 2MeSAMP or 53uM aspirin. Then, the platelet added in antagonist was co-incubated in 37℃ for 10 min. Adding 0.5U thrombin to activate the platelet for 5 min or 1 hour. 8%PFA in PHEM at a final concentration of 4%PFA was used to fix the remaining platelet for 15 min. Incubated antibodies of CD63 and vwf. The supernatant of activated platelet was collected for ELISA test.

Granule release testing

The supernatant of activated platelet was collected and centrifuged at 4℃ 1000×g for 15 min after stopping on the ice. Removing the supernatant to the Eppendorf tube performed PF4 and 5-HT analysis according to the instructions of the Elisa Kit or reserved it in -80℃.

Platelets observation and analyzation

The images were acquired from SIM. 9 raw images taken by nine different sinusoidal illumination patterns were reconstructed to one final image waiting for analyzing[12]. Sample was successively abrupted images under two different channel excitation light at 488nm (CD63) and 647 nm (vwf). Acquired SIM images could analyze the average diameter, average number of CD63 and vwf in single platelet, the average size of CD63 and vwf, mean area of CD63 and vwf per platelet and %area of CD63 and vwf of platelets by Image J plugins.

Data analysis

Data processing is anonymous. All the data was analysis by GraphPad Prism8.3.0. Unpaired, two-tailed student T-test was adopted to find differences between AIS patient and healthy control in platelet diameter, granule number, granule size, mean area of granule per platelet and %area of granule, if the data contented with normal distribution. Vwf average size was analyzed by two-tailed Mann Whitney test because of the data doesn't fit a normal distribution. P value < 0.05 of T-test was regarded as significant differences.

Ethical approval

The study had acquired ethic approval through Wuhan Forth Hospital and the approval number was Lun Shen Zi (KY2021-086-01). All research was performed in accordance with relevant guidelines and regulations. All the volunteers signed informed consent forms.

Data availability

All data generated or analyzed during this study are included in this published article (and its Supplementary Information files).

Acknowledgements

We thank all the volunteers for their participation in the study. The authors thank Huan Deng and Qian Jiang for assisting in laboratory equipment. And we thank all other staff at neurology department in Wuhan Puai Hospital for help collecting samples, as well as blood transfusion laboratory of Wuhan Blood Center for supplying equipment.

Author contributions

Bingxin Yang performed the experiment and edited the manuscript. Xifeng Wang contributed to the conception of study. Xiaoyu Hu contributed to analysis and manuscript preparation. Yao Xiao and Xueyu Xu helped perform the analysis with constructive discussion. Xiaomei Yu and Min Wang did the literature search. Honglian Luo helped image representation. Jun Li contributed to manuscript preparation and data analysis. Yan Ma provided technical and fund support. Wei Shen contributed to the conception of study and fund support. All authors read and approved the manuscript.

Funding

Fund for Knowledge Innovation of Wuhan Science and Technology Bureau (Grant No.2022020801010558). Fund for Special fund for Young Backbone talents of Wuhan Fourth Hospital. This work also supported by the Medical Research Project of Wuhan Municipal Health Commission (Grant No. WG14B13), the National Natural Science Foundation of China (Grant No. 31600692), and the Natural Science Foundation of Hubei Province (2017CFB406).

Competing interest

The author(s) declare no competing interests.

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No competing interests reported.

SupplementaryFigureS13.pdf

Changes of platelet morphology, ultrastructure and function in patients with acute ischemic stroke based on super- resolution microscopy

Status:

Version 1

Abstract

Figures

Introduction

Results

No significant changes of rest platelet morphology were observed in AIS patients by SIM

Discussion

Materials And Methods

Materials and reagents

Patients’ selection

Control group choosing

Platelet preparation

Granule release testing

Platelets observation and analyzation

Data analysis

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1